Products of manufacture comprising bacteriophages

ABSTRACT

Provided are compositions and methods for treating, ameliorating and preventing various infections, disorders and conditions in mammals, including genetically-predisposed and chronic disorders, where a microbial or bacterial flora is at least one causative or symptom-producing factor, where exemplary compositions are products of manufacture, a food, a drink, a nutraceutical, a dietary supplement, a formulation, a pharmaceutical or a pharmaceutical preparation comprising at least one or several of a plurality of isolated, or substantially purified bacteriophages or prophages, or bacteriophage subunits, a milk, a milk product, milk lipid, milk fat globule (MFG) macromolecule, a milk mucin, a milk glycolipid, a milk free glycan, a milk mucin-like glycoprotein, a milk protein, a milk sugar or lactose, a milk fat or butterfat, a milk vitamin. In alternative embodiment, provided are compositions and methods for treating, preventing or ameliorating an infection, for example, an infection in the gastrointestinal tract, or bowel.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers NIHR01: GM095384, and NIH R21: AI094534, awarded by the National Instituteof General Medical, National Institutes of Health (NIH), DHHS. Thegovernment has certain rights in the invention.

RELATED APPLICATIONS

This U.S. utility patent application is a national phase applicationclaiming benefit of priority under 35 U.S.C. § 371 to Patent ConventionTreaty (PCT) International Application Serial No: PCT/US2015/061409,filed Nov. 18, 2015, which claims benefit of priority under 35 U.S.C. §119(e) of U.S. provisional patent application Ser. No. 62/082,074, filedNov. 19, 2014, and U.S. Ser. No. 62/126,577, filed on Feb. 28, 2015. Theaforementioned applications are expressly incorporated herein byreference in their entirety and for all purposes.

TECHNICAL FIELD

This invention generally relates to medicine, infectious diseases,pharmacology and microbiology. In alternative embodiments, provided arecompositions and methods for treating, ameliorating and preventingvarious infections, disorders and conditions in mammals, includinggenetically-predisposed and chronic disorders, where a microbial orbacterial flora is at least one causative or symptom-producing factor,where exemplary compositions as provided herein are products ofmanufacture, a food, a drink, a nutraceutical, a formulation, apharmaceutical or a pharmaceutical preparation comprising at least oneor several of: a plurality of isolated, or substantially purifiedbacteriophages, or bacteriophage subunits, a milk, a milk product, milklipid, milk fat globule (MFG) macromolecule, a milk mucin, a milkglycolipid, a milk free glycan, a milk mucin-like glycoprotein, a milkprotein, a milk sugar or lactose, a milk fat or butterfat, a milkvitamin, or equivalents thereof, or a mixture thereof. In alternativeembodiment, provided are compositions and methods for treating,preventing or ameliorating an infection, for example, an infection inthe gastrointestinal tract, or bowel. In alternative embodiments,provided are compositions and methods using bacteriophage orbacteriophages subunits for targeting specific infectious agents orpathogens, for example, bacteria.

BACKGROUND

Mucosal surfaces are the primary zones where animals meet theirenvironment, and thus also the main points of entry for pathogenicmicroorganisms. The mucus layer is heavily colonized by bacterialsymbionts that provide additional genetic and metabolic potentials tothe host (1, 2). Bacterial symbionts are associated with a variety ofhost surfaces, providing beneficial goods and services, e.g., nutrients(3-6), bioluminescence (7, 8), and antibiotics (9, 10). The residentsymbiotic bacteria benefit from the available nutrients, as well as theopportunity for both vertical transmission and increased dissemination(11-13).

Within the mucus, the predominant macromolecules are the large (up to10⁶-10⁹ Da) mucin glycoproteins. The amino acid backbone of theseproteins incorporates tandem repeats of exposed hydrophobic regionsalternating with blocks with extensive O-linked glycosylation (14).Hundreds of variable, branched, negatively-charged glycan chains extend0.5-5 nm from the peptide core outward into the surrounding environment(14, 15). In addition to mucins, DNA, proteins and other cellular debrisare also present. Continual secretion and shedding of mucins maintains aprotective mucus layer from 10 to 700 μm thick, depending on species andbody location (16-19).

By offering both structure and nutrients, mucus layers commonly supporthigher bacterial concentrations than the surrounding environment (20,21). Secretions produced by the underlying epithelium influence thecomposition of this microbiota (22-24). Of necessity, hosts employ avariety of mechanisms to limit microbial colonization (23, 25-27). Wheninvaded by pathogens, the epithelium may respond by increased productionof anti-microbial agents, hypersecretion of mucin, or alteration ofmucin glycosylation patterns to subvert microbial attachment (28-30).

Also present in the mucus environment are bacteriophage (phage), themost common and diverse biological entities. As specific bacterialpredators, they increase microbial diversity through RedQueen/Kill-the-Winner dynamics (31, 32). Many phages instead establishsymbiotic relationships with their bacterial hosts through lysogeny. Asintegrated prophages they often express genes that increase host fitnessor virulence (33-35) and protect their host from lysis by relatedphages. As free phages they aid their host strain by killing competingstrains (36-38). Phages participate, along with their bacterial hosts,in tripartite symbioses with metazoans that affect metazoan fitness(39-42). However, no direct symbiotic interactions between phage andmetazoans are known.

Recently, Minot et al. showed that phages in the human gut encode apopulation of hypervariable proteins (43). For 29 hypervariable regions,evidence indicated that hypervariability was conferred by targetedmutagenesis through a reverse transcription mechanism (43, 44).Approximately half of these encoded proteins (14) possessed the C-typelectin fold previously found in the major tropism determinant protein atthe tip of the Bordetella phage BPP-1 tail fibers (45); six otherscontained Ig-like domains. These Ig-like proteins, similar to antibodiesand T-cell receptors, can accommodate large sequence variation (>10¹³potential alternatives) (46). Immunoglobulin-like (Ig-like) domains arealso displayed in the structural proteins of many phage (47, 48). Thatmost of these displayed Ig-like domains are dispensable for phage growthin the laboratory (44, 48) led to the hypothesis that they aidadsorption to their bacterial prey under environmental conditions (48).The possible role and function of these hypervariable proteins remain tobe clarified.

SUMMARY

In alternative embodiments, provided are products of manufacture, foodsor feeds, dietary supplements, drinks, nutraceuticals, formulations(e.g., infant formulations), pharmaceuticals and pharmaceuticalpreparations comprising a plurality of isolated, or substantiallypurified bacteriophages or prophages formulated with: (a) a milk,wherein optionally the milk is a human milk; (b) a milk productcomprising a milk lipid, a milk fat globule (MFG) macromolecule, a mucinfound in a milk (a milk mucin), a glycolipid found in a milk (a milkglycolipid), a free glycan found in a milk (a milk free glycan), amucin-like glycoprotein found in a milk (a milk mucin-likeglycoprotein), a milk protein (e.g., a casein, a casein micelle, or awhey, e.g., a beta-lactoglobulin or an alpha-lactalbumin), a milk sugaror a lactose, a milk fat or a butterfat (e.g., a saturated fatty acid,e.g., a Palmitic acid, a Myristic acid, a Stearic acid, a lower e.g.,less than 12 carbon, saturated fatty acids, a pentadecanoic acid, or aheptadecanoic acid; or, an unsaturated fatty acid, e.g., an Oleic acid,a Palmitoleic acid, a Linoleic acid or an alpha-Linolenic acid), a milkvitamin (e.g., water soluble vitamins thiamin (vitamin B1), riboflavin(vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5),vitamin B6 (pyridoxine), vitamin B12 (cobalamin), vitamin C (L-ascorbicacid) or folate) and fat soluble vitamin, e.g., a vitamin A, a vitamin D(e.g., a secosteroid, a calcidiol, a hydroxycholecalciferol or acholecalciferol), a vitamin E (e.g., a tocopherol or a tocotrienol), ora vitamin K (e.g., a phylloquinone, a phytomenadione, or aphytonadione)), or equivalents thereof, or a mixture thereof; (c) anisolated, or substantially purified: milk lipid, milk fat globule (MFG)macromolecule, mucin found in a milk (a milk mucin), glycolipid found ina milk (a milk glycolipid), free glycan found in a milk (a milk freeglycan), mucin-like glycoprotein found in a milk (a milk mucin-likeglycoprotein), milk protein, milk sugar or lactose, milk fat orbutterfat, milk vitamin, or equivalents thereof, or a mixture thereof;(d) a liposome or a micelle, a hydrogel (e.g., a biocompatiblecrosslinked degradable thiol-ene polymer) a dendrimer, a particle or amicroparticle (e.g., a biodegradable polymeric particle, e.g., apoly(lactic acid) (PLA), a poly(glycolic acid)—(PGA)—or apoly(lactic-co-glycolic acid) (PLGA)-comprising particle), a powder, ananostructure or a nanoparticle comprising a component of any of (a),(b) or (c); or (e) a poly(lactic-co-glycolic acid) (PLGA); a poly(lacticacid) (PLA); a poly(glycolic acid); a poly(vinyl alcohol)(PVA); apoly(ethylene glycol)(PEG); a poly(ethylene oxide); a poly(ethyleneoxide)-co-poly(propylene oxide) block copolymer; a polyoxamine; apolyanhydride; a polyorthoester; a poly(hydroxyl acids); apolydioxanone; a polycarbonate; a polyaminocarbonate; a poly(vinylpyrrolidone; a poly(ethyl oxazoline); a carboxymethyl cellulose; ahydroxyalkylated cellulose; a heparin sulfate, a chondroitin sulfate, aheparin, an alginate, a gelatin, a collagen, a albumin, a ovalbumin, orequivalents thereof, or a mixture thereof; or (f) any combination of theplurality of isolated, or substantially purified bacteriophages orprophages with (a), (b), (c), (d) and/or (e) thereof.

In alternative embodiments, provided are products of manufacture, foodsor feeds, drinks, nutraceuticals, dietary supplements, formulations(e.g., infant formulations), pharmaceuticals and pharmaceuticalpreparations comprising:

(a) a plurality of isolated, or substantially purified bacteriophages orprophages,

-   -   wherein optionally the prophage is an extrachromosomal plasmid        form, or is integrated into the genome of a microorganism,        optionally a bacteria, optionally a probiotic bacterium;

(b) a milk or a cream,

wherein optionally the milk is a mammalian or a human milk, andoptionally the milk is a skim milk or a whole (full) milk;

(c) a milk product, milk component or milk isolate comprising: a milklipid, a milk fat globule (MFG) macromolecule (including intact, e.g.,non-pasteurized) milk fat globules (MFGs), which comprise e.g., thecream fraction of milk and fat droplets which are stabilized by anexternal membrane derived mainly from the apical plasma membrane ofmammary secretory cells), a mucin found in a milk (a milk mucin), aglycolipid found in a milk (a milk glycolipid), a free glycan found in amilk (a milk free glycan), a mucin-like glycoprotein found in a milk (amilk mucin-like glycoprotein), a milk protein (e.g., a casein, a caseinmicelle, or a whey, e.g., a beta-lactoglobulin or an alpha-lactalbumin),a milk sugar or a lactose, a milk fat or a butterfat (e.g., a saturatedfatty acid, e.g., a Palmitic acid, a Myristic acid, a Stearic acid, alower e.g., less than 12 carbon, saturated fatty acids, a pentadecanoicacid, or a heptadecanoic acid; or, an unsaturated fatty acid, e.g., anOleic acid, a Palmitoleic acid, a Linoleic acid or an alpha-Linolenicacid), a milk vitamin (e.g., water soluble vitamins thiamin (vitaminB1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid(vitamin B5), vitamin B6 (pyridoxine), vitamin B12 (cobalamin), vitaminC (L-ascorbic acid) or folate) and fat soluble vitamin, e.g., a vitaminA, a vitamin D (e.g., a secosteroid, a calcidiol, ahydroxycholecalciferol or a cholecalciferol), a vitamin E (e.g., atocopherol or a tocotrienol), or a vitamin K (e.g., a phylloquinone, aphytomenadione, or a phytonadione)), or equivalents thereof, or amixture thereof;

(d) an isolated, or substantially purified: milk lipid, milk fat globule(MFG) macromolecule, mucin found in a milk (a milk mucin), glycolipidfound in a milk (a milk glycolipid), free glycan found in a milk (a milkfree glycan), mucin-like glycoprotein found in a milk (a milk mucin-likeglycoprotein), milk protein, milk sugar or lactose, milk fat orbutterfat, milk vitamin, or equivalents thereof, or a mixture thereof;

(e) a liposome or a micelle, a plasma membrane, or component of a plasmamembrane (optionally a bacterial plasma membrane), a hydrogel (e.g., abiocompatible crosslinked degradable thiol-ene polymer) a dendrimer, aparticle or a microparticle (e.g., a biodegradable polymeric particle,e.g., a poly(lactic acid) (PLA), a poly(glycolic acid)—(PGA)—or apoly(lactic-co-glycolic acid) (PLGA)-comprising particle), a powder, ananostructure or a nanoparticle comprising a component of any of (a),(b), (c) or (d),

wherein optionally the plasma membrane, or component of a plasmamembrane, is reconstituted as a globular particle of a specific sizerange, optionally having a size of between about: 1 nm to 100 nm, 100 nmto 1000 nm, 1 um to 100 um, or 100 um to 1000 um,

and optionally the plasma membrane component comprises a milk fatglobule membrane, another lipid bilayer, another lipid bilayercomprising and displaying a mucin or a negatively charged glycan;

(f) a poly(lactic-co-glycolic acid) (PLGA); a poly(lactic acid) (PLA); apoly(glycolic acid); a poly(vinyl alcohol)(PVA); a poly(ethyleneglycol)(PEG); a poly(ethylene oxide); a poly(ethyleneoxide)-co-poly(propylene oxide) block copolymer; a polyoxamine; apolyanhydride; a polyorthoester; a poly(hydroxyl acids); apolydioxanone; a polycarbonate; a polyaminocarbonate; a poly(vinylpyrrolidone; a poly(ethyl oxazoline); a carboxymethyl cellulose; ahydroxyalkylated cellulose; a heparin sulfate, a chondroitin sulfate, aheparin, an alginate, a gelatin, a collagen, a albumin, a ovalbumin, orequivalents thereof, or a mixture thereof;

(g) the combination of: (a) and (b): (a) and (c); (a) and (d); (a) and(e); (a) and (f); (a) and (g); (a), (b) and (e); (a), (b) and (f); (a),(c) and (e); (a), (c) and (f); (a), (d) and (e); or (a), (d) and (f); or

(h) any combination thereof,

wherein optionally the mucin comprises a membrane-tethered,membrane-integrated or secreted mucin, and optionally the mucincomprises a MUC1, MUC3A, MUC3B, MUC4, MUC10, MUC11, MUC12, MUC13, MUC14,MUC15, MUC16, MUC17, MUC18, MUC20, MUC21, MUC2, MUC5AC, MUC5B, MUC6,MUC7, MUC8, MUC9, MUC19,

and optionally the glycan is an O-linked glycosylated glycan, anoptionally a glycan with O-linked GalNAc or N-linked glycosylation, andoptionally a glycan as set forth in FIG. 28.

In alternative embodiments, the plurality of isolated, or substantiallypurified bacteriophages or prophages are selected from the groupconsisting of:

(a) a plurality of isolated, or substantially purified bacteriophages orprophages derived from a mammal, wherein optionally the mammal is human;

(b) a plurality of isolated, or substantially purified bacteriophages orprophages derived or isolated from milk of a mammal, wherein optionallythe mammal milk is a human milk;

(c) a plurality of isolated, or substantially purified bacteriophages orprophages from the order Caudovirales or Ligamenvirales;

(d) a plurality of isolated, or substantially purified bacteriophages orprophages from the family Myoviridae, Siphoviridae, Podoviridae,Lipothrixviridae, Rudiviridae, Ampullaviridae, Bicaudaviridae,Clavaviridae, Corticoviridae, Cystoviridae, Fuselloviridae,Globuloviridae, Guttaviridaelnoviridae, Leviviridae, Microviridae,Plasmaviridae or a combination thereof;

(e) a plurality of isolated, or substantially purified bacteriophages orprophages known or demonstrated to be toxic or lysogenic to a bacteria,or is bacteriocidal or bacteriostatic, or can treat, inhibit or preventan infection (optionally a microbial or a bacterial infection), whereinoptionally the bacteriophages or prophages are bacteriocidal orbacteriostatic to gram negative bacteria or gram positive bacteria,optionally the bacteria or infection is or is caused by an MSRAinfection, a Staphylococcus, a Staphylococcus aureus, a Clostridium, ora Clostridium difficile, Escherichia coli, a Shigella, a Salmonella, aCampylobacter, a Cholerae, a Bacillus, or a Yersinia;

(f) a plurality of isolated, or substantially purified bacteriophages orprophages made by a process comprising: screening a plurality ofbacteriophages or prophages for bacteriocidal or bacteriostaticproperties against a bacteria of interest, and selecting thebacteriophages or prophages having a lysogenic or a bacteriocidal orbacteriostatic activity;

(g) a plurality of isolated or substantially purified temperate phage,optionally a phage or prophage that are either integrated or excisedfrom a bacteria, optionally a probiotic bacteria; or

(f) any combination thereof.

In alternative embodiments: (a) the bacteriophage or prophage areisolated or substantially purified based on the bacteriophage orprophage having the following properties; or, the bacteriophage orprophage are characterized as having the following properties: having acesium chloride density between 1.5 to 1.7 g/mL, being resistant tolysis by chloroform, having a particle size between about 0.01 and 0.2μm, between about 0.005 and 0.5 μm or between about 0.001 and 0.8 μm,and being detectable via dsDNA fluorescence stain; or, (b) thebacteriophage or prophage are formulated per dose, or per serving, orper unit dosage at, or at a total daily dose of: between about 10(1) (or10¹) and 10(20) plaque-forming units (PFUs), or between about 10(3) and10(17) PFUs, or between about 10(5) and 10(12) PFUs, or between about10(7) and 10(9) PFUs.

In alternative embodiments, the products of manufacture, foods or feeds,dietary supplements, drinks, nutraceuticals, formulations (e.g., infantformulations), pharmaceuticals and pharmaceutical preparations furthercomprise: an additional active agent or pharmaceutical or pharmaceuticalpreparation, wherein optionally the additional pharmaceutical orpharmaceutical preparation comprises an antibiotic, and optionally theadditional active agent comprises a microorganism, optionally a bacteriaor a yeast, optionally a probiotic bacteria, and optionally theadditional active agent comprises a probiotic, a prebiotic, apostbiotic, or a synbiotic, and optionally the prebiotic comprises anondigestible oligosaccharide, optionally an inulin, a fructan, axylose, a maltose, a mannose, or a fructooligosaccharide, and optionallythe probiotic bacteria comprise: a Bacillus or a Lactobacillus; or, aBacillus coagulans, a Lactobacillus acidophilus, a Lactobacillusplantarum or a Lactobacillus rhamnosus, and optionally the yeastcomprises a Saccharomyces; or a Saccharomyces boulardii.

In alternative embodiments, the products of manufacture, foods or feeds,dietary supplements, drinks, nutraceuticals, formulations (e.g., infantformulations), pharmaceuticals and pharmaceutical preparations aremanufactured, formulated, dosaged and/or packaged for administration invivo; or for enteral or parenteral administration, or for ophthalmic,topical, oral, intranasal, intrarectal, intravaginal, intravenous,intra-peritoneal, intraocular, intra-placental, intra-bladder,cutaneous, intravenous (IV), intramuscular (IM), intrathecal,subcutaneous (SC), intracerebral, epidural, intracranial or rectaladministration, or by inhalation.

In alternative embodiments, the products of manufacture, foods or feeds,dietary supplements, drinks, nutraceuticals, formulations (e.g., infantformulations), pharmaceuticals and pharmaceutical preparations areformulated, dosaged, manufactured as or placed or formulated into: (a) aparticle, a nanoparticle, a liposome, a tablet, a pill, a capsule, agel, a geltab, a liquid, a powder, a suspension, a syrup, an emulsion, alotion, an ointment, an aerosol, a spray, a lozenge, an ophthalmicpreparation, an aqueous or a sterile or an injectable solution, a patch(optionally a transdermal patch or a medicated adhesive patch), animplant, a dietary supplement, an ice cream, an ice, a yogurt, a cheese,an infant formula or infant dietary supplement, a pasteurized milk ormilk product or milk-comprising product; or, (b) a veterinaryformulations or feed, or as dietary supplements.

In alternative embodiments, the products of manufacture, foods or feeds,dietary supplements, drinks, nutraceuticals, formulations (e.g., infantformulations), pharmaceuticals and pharmaceutical preparations furthercomprise, or have added to:

a pharmaceutically acceptable excipient.

a flavoring or a sweetening agent, an aspartamine, a stevia, monk fruit,a sucralose, a saccharin, a cyclamate, a xylitol, a vanilla, anartificial vanilla or chocolate or strawberry flavor, an artificialchocolate essence, or a mixture or combination thereof.

a preservative, a benzoic acid, a potassium sorbate.

at least one probiotic or prebiotic, wherein optionally the prebioticcomprises an inulin, lactulose, extracts of artichoke, chicory root,oats, barley, various legumes, garlic, kale, beans or flacks or an herb,

at least one congealing agent, wherein optionally the congealing agentcomprises an arrowroot or a plant starch, a powdered flour, a powderedpotato or potato starch, an absorbant polymer, an ABSORBABLE MODIFIEDPOLYMER® (AMP®), (EndoClot, Santa Clara, Calif.), and/or a corn flour ora corn starch.

at least one an anti-inflammatory agent, wherein optionally theinflammatory agent comprises or is an NSAID, a 4 or a5-amino-salicylate, an olsalazine, a mesalazine, a sulfasalazine and/ora balsalazide or an equivalent thereof or a combination thereof.

an additive selected from one or more of a saline, a media, a defoamingagent, a surfactant agent, a lubricant, an acid neutralizer, a marker, acell marker, a drug, an antibiotic, a contrast agent, a dispersal agent,a buffer or a buffering agent, a sweetening agent, a debittering agent,a flavoring agent, a pH stabilizer, an acidifying agent, a preservative,a desweetening agent and/or coloring agent, vitamin, mineral and/ordietary supplement, or a prebiotic nutrient,

and optionally the buffer or a buffering agent or the pharmaceuticallyacceptable excipient comprises an inorganic salt, a citric acid, asodium chloride, a potassium chloride, a sodium sulfate, a potassiumnitrate, a sodium phosphate monobasic, a sodium phosphate dibasic orcombinations thereof,

and optionally the antacid comprises a calcium carbonate, a magnesiumhydroxide, a magnesium oxide, a magnesium carbonate, an aluminumhydroxide, a sodium bicarbonate or a dihydroxyaluminum sodium carbonate;or

any combination thereof.

In alternative embodiments, the products of manufacture, foods or feeds,dietary supplements, drinks, nutraceuticals, formulations (e.g., infantformulations), pharmaceuticals and pharmaceutical preparations aremanufactured, processed, isolated, treated, formulated or prepared:

(a) as a delayed or gradual enteric release composition or formulation,

and optionally the formulation comprises a gastro-resistant coatingdesigned to dissolve at a pH of 7 in the terminal ileum, e.g., an activeingredient is coated with an acrylic based resin or equivalent, e.g., apoly(meth)acrylate, e.g. a methacrylic acid copolymer B, NF, such asEUDRAGIT S™ (Evonik Industries AG, Essen, Germany), which dissolves atpH 7 or greater, e.g., comprises a multimatrix (MMX) formulation; or

(b) by treating the milk, the cream, the milk product, the milkcomponent or the milk isolate, or a component of (1)(a), (1)(b), (1)(c),(1)(d), (1)(e) or (1)(f), to a process selected from the groupconsisting of: aeration, agitation, stirring, pumping, shaking, rocking,centrifugation, temperature, incubation, drying, ageing, pasteurizing,homogenizing, coaggulation, chemical addition, bacterial growth, flowcytometry, particle size selection, fluorescent tagging or labelling,rheology, microscopy, micromanipulation, microfluidics, densitygradients, gel electrophoresis, column filtration and any combinationthereof.

In alternative embodiments, the milk, the cream, the milk product, themilk component or the milk isolate is isolated, processed, manufactured,or derived from:

(a) a mammal ranging in age from between about 1 to 3 years, 3 to 5years, or 5 to 10 years of age;

(b) a mammal ranging in age from between about 1 to 30 days postnatal,30 to 90 days postnatal, or 90 to 360 postnatal;

(c) a mammal during a specific season or month of the year;

(d) a mammal fed on a diet or feed: high in fat, protein and/orcarbohydrate content;

(e) a mammal bred to increase a specific component of milk or cream; or

(f) a specific breed of mammal or individual animal that is infected,diseased, or suffering from a mastitis, clinical and non-clinical.

In alternative embodiments, the milk, the cream, the milk product, themilk component or the milk isolate comprises, or consists of, componentshaving a particle size between about: 0.1 nm to 10.0 nm, 1 nm to 100 nm,100 nm to 1000 nm, 1 um to 100 um, or 100 um to 1000 um.

In alternative embodiments, the milk, the cream, the milk product, themilk component or the milk isolate comprises between about 0.01% to 1%,1% to 20%, or 20% to 100% of the food or a feed, drink, nutraceutical,formulation, pharmaceutical or pharmaceutical preparation.

In alternative embodiments, provided are delivery vehicles, products ofmanufacture, containers, syringes, devices or implants, comprising:foods or feeds, drinks, dietary supplements, nutraceuticals,formulations, freeze-dried compositions, an infant formula,pharmaceuticals or pharmaceutical preparations comprising, consisting ofor having contained therein or being packaged or stored therein one ormore or a plurality of compositions as provided herein, e.g., productsof manufacture, foods or feeds, dietary supplements, drinks,nutraceuticals, formulations (e.g., infant formulations),pharmaceuticals and pharmaceutical preparations as described herein.

In alternative embodiments, provided are products of manufacture,liquids, suspensions, gels, geltabs, a semisolid, a tablet, a sachet, alozenge or a capsule, a freeze-dried composition, an infant formula, oran enteral or parenteral formulation, comprising: food or a feed, drink,nutraceutical, formulation, pharmaceutical or pharmaceutical preparationas provided herein, e.g., one or more or a plurality of compositions asdescribed herein.

In alternative embodiments, provided are methods for:

-   -   treating, ameliorating and preventing a microbial or a bacterial        infection,    -   increasing or decreasing an effective dose of a probiotic, a        prebiotic, a postbiotic or a synbiotic;    -   increasing or decreasing efficacy of a probiotic, a prebiotic, a        postbiotic or a synbiotic attaching to mucosal surfaces;    -   increasing or decreasing access of a probiotic, a prebiotic, a        postbiotic or a synbiotic to mucosal surfaces;    -   increasing or decreasing chance of an enteral or        gastrointestinal pathogenic infection or disease; or    -   increasing or decreasing resistance of a probiotic, a prebiotic,        a postbiotic or a synbiotic to temperature, pH, mechanical        stress, osmotic stress and/or gastrointestinal enzymes,

comprising administering, feeding or applying to an individual in needthereof:

(a) a product of manufacture, food or a feed, drink, nutraceutical,formulation, pharmaceutical or pharmaceutical preparation as providedherein, or as described herein;

(b) a delivery vehicle, a product of manufacture, a container, asyringe, device or an implant as provided herein, or as describedherein; or

(c) liquid, a suspension, a gel, a geltab, a semisolid, a tablet, asachet, a lozenge or a capsule, a freeze-dried composition, an infantformula, or an enteral or parenteral formulation, comprising: food or afeed, drink, nutraceutical, formulation as provided herein, or asdescribed herein,

wherein optionally the individual is a mammal, and optionally the mammalis a human, or a human infant,

and optionally an antacid or a buffer or buffering agent or apharmaceutically acceptable excipient is administered before, during orafter, or before and during, administration of the product ofmanufacture, food or a feed, drink, nutraceutical, formulation,pharmaceutical or pharmaceutical preparation of (a),

and optionally sufficient amount of antacid, buffer or buffering agentis administered (optionally before, during or after, or before andduring, administration) to raise the pH of the stomach in the individualto between about 2.5 and 7, or between about 3 and 6.5, or to about 5.0,5.5, 6.0, 6.5, 6.8 or 7.0 (optionally these pH values reached before,during or after, or before and during, administration),

and optionally the buffer or a buffering agent or the pharmaceuticallyacceptable excipient comprises an inorganic salt, a citric acid, asodium chloride, a potassium chloride, a sodium sulfate, a potassiumnitrate, a sodium phosphate monobasic, a sodium phosphate dibasic orcombinations thereof,

and optionally the antacid comprises a calcium carbonate, a magnesiumhydroxide, a magnesium oxide, a magnesium carbonate, an aluminumhydroxide, a sodium bicarbonate or a dihydroxyaluminum sodium carbonate.

In alternative embodiments, provided are Uses of:

(a) a product of manufacture, a food or a feed, a drink, anutraceutical, a formulation, a pharmaceutical or a pharmaceuticalpreparation as provided herein, or as described herein;

(b) a delivery vehicle, a product of manufacture, a container, asyringe, device or an implant as provided herein, or as describedherein; or

(c) a liquid, a suspension, a gel, a geltab, a semisolid, a tablet, asachet, a lozenge or a capsule, a freeze-dried composition, an infantformula, or an enteral or parenteral formulation, comprising: food or afeed, drink, nutraceutical, formulation as provided herein, or asdescribed herein,

for:

-   -   treating, ameliorating and preventing a microbial or a bacterial        infection,    -   increasing or decreasing an effective dose of a probiotic, a        prebiotic, a postbiotic or a synbiotic;    -   increasing or decreasing efficacy of a probiotic, a prebiotic, a        postbiotic or a synbiotic attaching to mucosal surfaces;    -   increasing or decreasing access of a probiotic, a prebiotic, a        postbiotic or a synbiotic to mucosal surfaces;    -   increasing or decreasing chance of an enteral or        gastrointestinal pathogenic infection or disease; or    -   increasing or decreasing resistance of a probiotic, a prebiotic,        a postbiotic or a synbiotic to temperature, pH, mechanical        stress, osmotic stress and/or gastrointestinal enzymes.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments asprovided herein and are not meant to limit the scope of the invention asencompassed by the claims.

FIG. 1a , FIG. 1b , FIG. 1c graphically illustrate data showing theeffect of Phage adhering to the mucin glycoprotein in mucus layers: FIG.1a , Phage-to-bacteria ratio (PBR) for diverse mucosal surfaces and theadjacent environment. On average, PBRs for mucosal surfaces were4.4-fold greater than for the adjacent environment (n=9, t=4.719***P=0.0002, unpaired t test); FIG. 1b , Phage adherence to TC cellmonolayers, with and without surface mucus (unpaired t tests). (left)Non-mucus producing Huh-7 liver hepatocyte cells and mucus-producing T84colon epithelial cells (n>18, t=8.366, ****P<0.0001); (center)mucus-producing A549 lung epithelial cells with and without treatmentwith NAC, a mucolytic agent (n>40, t=9.561, ****P<0.0001); (right)mucus-producing shRNA control A549 cells (shControl) and mucus-knockdown(MUC) A549 cells (n>37, t=7.673, ****P<0.0001); FIG. 1c , Phageadherence to agar plates coated with mucin, DNA, or protein and anuncoated agar control (n=12, t=5.306, ****P<0.0001, unpaired t test); asfurther discussed in Example 1, below.

FIG. 2a and FIG. 2b graphically illustrate data showing the effect ofphage adsorption on bacterial infection of epithelial cells: FIG. 2a ,Bacterial attachment to mucus producing (T84 and A549) and non-mucusproducing (Huh-7, MUC⁻) TC cells, with and without phage pretreatment.Phage pretreatment significantly decreased subsequent bacterialadherence to mucus-producing TC cell lines (T84; n>30, t=32.05,****P<0.0001, A549; n>30, t=36.85, ****P<0.0001, unpaired t tests). Lessdramatic shifts were seen for non-mucus producing cells (Huh-7; n>30,t=2.72, **P=0.0098, MUC⁻; n>30, t=3.52, ***P=0.0007, unpaired t tests).FIG. 2b , Mortality of mucus-producing (A549) and mucus knockdown (MUC⁻)A549 lung epithelial cells following overnight incubation with E. coli(ns=not significant). Phage pretreatment completely protectedmucus-producing A549 cells from bacterial challenge (n=12, ****P<0.0001,Tukey's one-way ANOVA), while phage comparatively protected MUC⁻ cells3.1-fold less (n=12, *P=0.0181); as further discussed in Example 1,below.

FIG. 3a , FIG. 3b , FIG. 3c graphically illustrate data showing theeffect of Hoc protein on phage-mucin interactions. FIG. 3a , Adherenceof hoc⁺ and hoc⁻ T4 phage to agar coated with mucin, DNA, or proteinversus plain agar controls (n>11, t=3.977, ***P=0.0007, unpaired ttest). FIG. 3b , Competitive adherence of hoc⁺ and hoc⁻ T4 phage in0%-5% mucin solutions that were washed over mucus-producing A549 cells(n=25 per sample). FIG. 3c , Diffusion of fluorescence-labeled hoc⁺(left) and hoc⁻ (right) T4 phage in buffer and 1% mucin as determined bymultiple particle tracking. Mucin hindered diffusion of hoc⁺ T4 phagebut not hoc⁻ phage (ten analyses per sample, trajectories of n>100particles for each analysis); as further discussed in Example 1, below.

FIG. 4a and FIG. 4b graphically and schematically illustrate datashowing Hoc-mediated glycan binding and Hoc-related phylogeny: FIG. 4a ,Phylogenetic tree of sequences from viral metagenomes with high sequencehomology to Ig-like domains; many of these homologs are frommucus-associated environments (e.g., human feces, sputum); also includedare T4 Hoc and hypervariable Ig-like domains previously obtained by deepsequencing of phage from the human gut (7). Scale bar represents anestimated 0.5 amino acid substitutions per site; FIG. 4b , Binding offluorescence stained hoc⁺ and hoc⁻ T4 phage to a microarray of 610mammalian glycans; Normalized relative fluorescence units (RFU) werecalculated from mean fluorescence minus background binding; as furtherdiscussed in Example 1, below.

FIG. 5 schematically illustrates the so-called “Bacteriophage Adherenceto Mucus (BAM) model”: 1) Mucus is produced and secreted by theunderlying epithelium. 2) Phage interact weakly with variable glycanresidues displayed on mucin glycoproteins, via variable capsid proteins(e.g., Ig-like domains). 3) Phage adherence creates an antimicrobiallayer that reduces bacterial attachment to and colonization of themucus, which in turn lessens epithelium cell death. 4) Since phage aremore likely to encounter bacterial hosts in the mucus, they are underpositive selection for capsid proteins that enable them to maintain inthe mucus layer; thus phage rapidly adapt to variable mucus glycans. 5)Continual sloughing of the outer mucus provides a dynamic mucosalenvironment; as further discussed in Example 1, below.

FIG. 6 illustrates a table showing the composition of a breast milk.

FIG. 7A, FIG. 7B and FIG. 7C schematically illustrate (FIG. 7A) and byillustrating images (FIG. 7B and FIG. 7C) describe and illustratecompounds found in breast milk.

FIG. 8 illustrates a table showing and describing how breast milksamples were collected and from whom, to analyze Virus-Like Particles(VLPs) in human breast milk to characterize a human breast milk viromepopulation, for analysis as shown in FIG. 9 and FIG. 10.

FIG. 9 graphically illustrates quantification of VLPs from human breastmilk.

FIG. 10 illustrates a table showing how the sequencing of the VLPscollected from human breast milk as illustrated in FIG. 9 was done.

FIG. 11 illustrates and describes possible avenues of transmission ofphage from human breast milk to an infant gut epithelium.

FIG. 12 schematically illustrates how breast milk oligosaccharides bindto bacteria, which can be pathogens, and this binding preventing thebacteria from attaching to gut cells of the gut cell wall, and that theoligosaccharides also can directly bind to the cell wall and alter whatcell receptors are displayed in the gut lumen.

FIG. 13 schematically illustrates and describes an experimental setup todetermine and quantify if milk (breast milk oligosaccharides) bind to(or associate with after washing) phage and/or adheres to cultured gutepithelial cells, and illustrates an image of phage adhering to anepithelial cell, in particular, the mucous-producing adherent culturedLS513 epithelial cell.

FIG. 14 graphically illustrates quantification of phage adherence toadherent cultured LS513 epithelial cells using the protocol of FIG. 13,where T4 phage were mixed with milk, buffer or formula, added to thecultured adherent cells, incubated, the cells then washed, and theadherent cells quantified by plaque forming units (PFU), and the datashows that there is a greater amount of phage binding to (or associatedwith after washing) adherent cells in the milk fraction as compared tothe formula or buffer fractions.

FIG. 15 illustrates a table describing from whom breast milk sample wascollected to analyze phage adherence to human breast milk and gutepithelium.

FIG. 16 graphically illustrates quantification of phage adherence toadherent cultured LS513 epithelial cells as for FIG. 13 and FIG. 14, andshows that there is a greater amount of phage binding to (or associatedwith after washing) the LS513 epithelial cells as compared to formula.

FIG. 17A and FIG. 17B graphically illustrate quantification of bacteriato adherent cultured LS513 epithelial cells, and the data shows: FIG.17A data illustrates that in the “bacteria/no phage” sample there arefewer bacteria adherent to (associated with after washing) the LS513epithelial cells in the milk fraction versus the formula fraction; andFIG. 17B illustrates that in the “bacteria/with phage” sample there areeven fewer bacteria adherent to (associated with after washing) theLS513 epithelial cells in the milk fraction versus the formula fraction,demonstrating that the “phage and milk” fraction is most effective inreducing bacterial adherence to the epithelial cells.

FIG. 18 illustrates an image, and describes, a milk fat globule (MFG)containing a triglyceride core coated by a lipid bilayer that displaysmucins (as indicated by the arrow), and that the MFG is covered orlayered by negatively charged mucins, and notes that formula does notcontain mucin-comprising MFGs, thus suggesting that the mucin componentof the MFG of milk may be responsible for increasing the effectivenessof the “phage and milk” fraction in preventing bacterial adherence tothe LS513 epithelial cells as found in the study of FIG. 17.

FIG. 19 illustrates an image, and describes, MFG separation from rawbreast milk by low centrifugation, the illustrated figure showing anMFG-enriched “cream layer” and an MFG-depleted “skim milk” layer.

FIG. 20A and FIG. 20B graphically illustrates how the MFG as isolated inFIG. 19 affects the adherence of phage in two different human milksamples (FIG. 20A and FIG. 20B, see FIG. 15), where the datademonstrates that the MFG (mixed with raw milk) is better than (FIG.20A) or is at least comparable to (FIG. 20B) raw and skim milk alone (noMFG), as compared to formula, in facilitating phage adherence to LS513epithelial cells (where T4 phage at 10⁷/ml was mixed with eitherformula, raw breast milk, skim milk or varying ratios of isolated MFGand raw milk; the mix layered onto the LS513 epithelial cells for 30min, 37° C., washed and adherent phage quantified by PFU.

FIG. 21 illustrates an image, and describes, how the negatively chargedmucins of breast milk MFGs facilitate the penetration of phage into anepithelial cell surface mucin layer.

FIG. 22 graphically illustrates quantification of phage adherent to anepithelial cell surface in five different human breast milk samples (seeFIG. 15) and formula, where either wild type (WT) or hoc⁻ T4 phage at10⁷/ml were mixed with breast milk for 30 min, 37° C., washed 3 times,the cells harvested and adherent phage quantified by PFU (as discussedbelow, Hoc structural proteins displayed on the T4 phage capsid interactwith mucin, thereby slowing phage diffusion in mucus and increasingphage mucosal residence time).

FIG. 23A, FIG. 23B, FIG. 23C, FIG. 23D, FIG. 23E, FIG. 23F and FIG. 23G(or FIG. 1, of Example 2) schematically and graphically illustrates anexemplary microfluidic device (chip) designed to simulate a life-like invitro mucosal surface, and their function, respectively. FIG. 23A)Schematic of chip design and measurements. FIG. 23B) Single chip bondedto a glass microscope slide with microfluidic tubing attached to in andout ports. FIG. 23C) Mucus-producing lung tissue cultured cells seededinto main channel. FIG. 23D) Cells in main after seven days of fluidflow and growth. E) Multiplex syringe pump running nine chipssimultaneously. FIG. 23F) Phage therapy assay with phage and bacterialcounts taken from the mucosal surface. FIG. 23G) Phage detachment ratesfrom mucosal surface over a six hour period, dashed line indicates the 1hr wash time point used in phage therapy assay; as further discussed inExample 2, below.

FIG. 24A and FIG. 24B (or FIG. 2, of Example 2) graphically illustrates:FIG. 24A) Mucus-adherent T4 wt and non-adherent T4 hoc⁻ phage diffusionconstants (μm²/s) in 0% (buffer), 0.2%, 1%, and 5% mucin solutions(wt/vol). FIG. 24B) Alpha value (α) measurements of T4 wt and T4 hoc⁻phage in 0% (buffer), 0.2%, 1%, and 5% mucin solutions (wt/vol).Brownian diffusion α≈1, subdiffusion α<1; as further discussed inExample 2, below.

FIG. 25 (or FIG. 3, of Example 2) graphically illustrates:Ensemble-averaged mean square displacement (MSD) (μm²) of mucus-adherentT4 wt and non-adherent T4 hoc⁻ phage in 0% (buffer), 0.2%, 1%, and 5%mucin solutions (wt/vol). Lines indicate best fit and alpha value (α)are the measurement of slope; as further discussed in Example 2, below.

FIG. 26A and FIG. 26B (or FIG. 4, of Example 2) graphically illustrates:Adsorption of relative phage (2×10⁵ mL⁻¹) and E. coli host (1×10⁷ mL⁻¹)over a 10 min period, theoretical calculations used the T4 adsorptionconstant (k)=2.4×10−9 cm³/min. FIG. 26A) T4 wt phage adsorption incontrol (0%) and 1% mucin (wt/vol) solutions, with k=2.3×10⁹ and 4.7×10⁹cm³/min, respectively. FIG. 26B) T4 hoc⁻ phage adsorption in control(0%) and 1% mucin (wt/vol) solutions, with k=2.1×10⁹ and 2.6×10⁹cm³/min, respectively; as further discussed in Example 2, below.

FIG. 27A and FIG. 27B (or FIGS. 5A and B, of Example 2) schematicallyand illustrates phage subdiffusion in a mucus network, and the phages'physical relationship to the exterior of a bacteria; as furtherdiscussed in Example 2, below.

FIG. 28 illustrates exemplary glycans that can be used to practiceembodiments as provided herein; as further discussed in Example 2,below.

Like reference symbols in the various drawings indicate like elements.

Reference will now be made in detail to various exemplary embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings. The following detailed description is provided to give thereader a better understanding of certain details of aspects andembodiments of the invention, and should not be interpreted as alimitation on the scope of the invention.

DETAILED DESCRIPTION

In alternative embodiments, provided are compositions and methods fortreating, ameliorating and preventing various infections, disorders andconditions in mammals, including genetically-predisposed and chronicdisorders, where a microbial or bacterial flora is at least onecausative or symptom-producing factor.

In alternative embodiment, provided herein are compositions and methodsfor treating, preventing or ameliorating conditions or infection causedby a microbe or a bacteria susceptible to lysis by a bacteriophage, orthe bacteriophage has a microbiocidal, a bacteriocidal or bacteriostaticeffect on the microbe or bacteria. In alternative embodiments, providedare products of manufacture, a food, a drink, a nutraceutical, aformulation, a pharmaceutical or a pharmaceutical preparation comprisinga plurality of isolated, or substantially purified bacteriophages, orbacteriophage subunits, formulated with a milk, a milk product, milklipid, milk fat globule (MFG) macromolecule, a milk mucin, a milkglycolipid, a milk free glycan, a milk mucin-like glycoprotein, a milkprotein, a milk sugar or lactose, a milk fat or butterfat, a milkvitamin, or equivalents thereof, or a mixture thereof.

In alternative embodiment, provided herein are compositions and methodsfor treating, preventing or ameliorating an infection, for example, aninfection in the gastrointestinal tract, or bowel, or any condition ordisease caused or exacerbated by a microbe in the gastrointestinaltract, e.g., a bacterial infection. In alternative embodiment,compositions and methods as provided herein are used to target a microbeor a bacteria that is pathogenic, or is associated with or completely orpartially causative of an infection or a condition, e.g., a colitis,obesity, diabetes, an autism and the like. In alternative embodiment,compositions and methods as provided herein are designed to target aparticular microbe or bacteria by selecting a particular bacteriophagethat has a microbiocidal, a bacteriocidal or bacteriostatic effect onthe targeted microbe or bacteria. In alternative embodiments,compositions and methods as provided herein comprise use of abacteriophage specific for, or a bacteriophage designed or constructedto be (e.g., by recombinant technology) specific for, or a bacteriophagesubunit responsible for specifically targeting, a particular infectiousagent or pathogen, a microbe or a bacteria that is pathogenic, or isassociated with or completely or partially causative of an infection ora condition, for example, bacteria.

In alternative embodiments, provided are compositions, e.g., a drugdelivery agent, a liposome or a micelle, a hydrogel, a dendrimer, aparticle or a microparticle, a powder, a nanostructure or ananoparticle, capable of targeting a specific microbe or bacteria, wherein alternative embodiments the specific targeting is effected byincorporation of a component of a bacteriophage specific for, or abacteriophage designed or constructed to be (e.g., by recombinanttechnology) specific for, or a bacteriophage subunit responsible forspecifically targeting, the specific microbe or bacteria, which can be aparticular infectious agent or pathogen, a microbe or a bacteria that ispathogenic, or is associated with or completely or partially causativeof an infection or a condition, for example, bacteria.

Milk Products, Isolates, Components

In alternative embodiments, provided are compositions, and methods ofusing them, comprising (or comprising use of) a milk, a cream, or a milkproduct, component or isolate. In alternative embodiments, a milk, acream, a milk product, milk component or milk isolate can comprise: amilk lipid, a milk fat globule (MFG) macromolecule, a mucin found in amilk (a milk mucin), a glycolipid found in a milk (a milk glycolipid), afree glycan found in a milk (a milk free glycan), a mucin-likeglycoprotein found in a milk (a milk mucin-like glycoprotein), a milkprotein (e.g., a casein, a casein micelle, or a whey, e.g., abeta-lactoglobulin or an alpha-lactalbumin), a milk sugar or a lactose,a milk fat or a butterfat. In alternative embodiments, a milk, a cream,a milk product, milk component or milk isolate can comprise: milk fatglobule (MFG) macromolecule, mucin found in a milk (a milk mucin),glycolipid found in a milk (a milk glycolipid), free glycan found in amilk (a milk free glycan), mucin-like glycoprotein found in a milk (amilk mucin-like glycoprotein), milk protein, milk sugar or lactose, milkfat or butterfat, milk vitamin, or equivalents thereof, or a mixturethereof.

Any method known in the art can be used to generate, process or make, amilk, a cream, or a milk product, component or isolate used to practiceembodiments as provided herein. For example, any known method or processused to process milk, e.g., breast milk, into its structural componentsor fractions, including full milk, skim milk, milk fat content rangingfrom 0.1% to 100%, cream, can be used, including methods and processesthat do not use heat, excessive heat, pasteurization and/or shredding,shearing or fragmenting of milk components such as milk fat globules,milk fat globule (MFG) macromolecules, or milk fat membranes.

For example, methods as described in WO/2001/035760 A1 can be used forthe bulk preparation of milk fat globule membranes. Alternatively,methods as described in US pat app publication nos. 2012 0321600 A1 or2014 0105875 A1, can be used for to isolating and/or preparing milk fatglobule (MFG) membranes and MFG components. Alternatively, methods andprocesses as described in US patents (USPN) or application publicationscan be used, for example: U.S. Pat. No. 5,861,491, which describesmethods for isolating and preparing macromolecular species present inthe milk, e.g., hydrophobic interaction chromatography; U.S. Pat. No.6,444,247, which describes preparation of milk fat globule membranes intablet or capsule form; U.S. Pat. No. 4,994,496, which describes use ofmilk fat globules as carriers for drugs; U.S. Pat. No. 4,997,668, whichdescribes production of low-cholesterol milk fat by solvent extraction;U.S. Pat. No. 8,846,604, which describes making lipid compositionscomprising phospholipids having a high docosahexaenoic acid (DHA)content; U.S. Pat. No. 8,012,509, which describes various milk fractionsand milk preparations; US pat app pub no 20140255539 and 20140255537,which describe making structured fat globules from an enriched lipidfraction derived from milk; US pat app pub no 20140093554 and20110300204 which describe making nutritional compositions for infantsand/or toddlers comprising a lipid component which has a large lipidglobule size; US pat app pub no 20100068293, describing making milkingredients enriched in polar lipids such as phospholipids and insphingolipids; US pat app pub nos 20140127350 and 20060150916 whichdescribe separating components from milk. Additionally, methodsdescribed in WO/2001/035760 A1, teaching bulk preparation methods forisolating milk fat globule membranes from milk, can be used.

Probiotics, Postbiotics, Prebiotics, and Synbiotics

In alternative embodiments, additives that are also included in acomposition as provided herein (e.g., a product of manufacture, food,drink, nutraceutical, formulation, pharmaceutical or pharmaceuticalpreparation), or a composition used to practice embodiments as providedherein, includes one or more prebiotics, postbiotics, probiotics orsynbiotics, including for example: inulin, lactulose, extracts ofartichoke, chicory root, oats, barley, various legumes, garlic, kale,beans or flacks and at times prebiotics may include herbs.

Also, in alternative embodiments, provided herein are uses and methodsfor: increasing or decreasing an effective dose of a probiotic, aprebiotic, a postbiotic or a synbiotic; increasing or decreasingefficacy of a probiotic, a prebiotic, a postbiotic or a synbioticattaching to mucosal surfaces; increasing or decreasing access of aprobiotic, a prebiotic, a postbiotic or a synbiotic to mucosal surfaces;or, increasing or decreasing resistance of a probiotic, a prebiotic, apostbiotic or a synbiotic to temperature, pH, mechanical stress, osmoticstress and/or gastrointestinal enzymes.

In alternative embodiments, additives may include beneficial (e.g.,prebiotics, postbiotics, probiotics or synbiotics) flora components suchas Bacteroidetes, Firmicutes, Bacillus (e.g., Bacillus thurigiensis) orany combination thereof. In alternative embodiments, cultured componentsare back to the flora to fortify or expand specific genus or species,e.g., Bacteroidetes, Firmicutes, Bacillus or Bacillus thurigiensis. Inalternative embodiments, prebiotics comprises a nondigestibleoligosaccharide, optionally an inulin, a fructan, a xylose, a maltose, amannose, or a fructooligosaccharide. In alternative embodiments,probiotic bacteria comprise: a Bacillus or a Lactobacillus; or, aBacillus coagulans, a Lactobacillus acidophilus, a Lactobacillusplantarum or a Lactobacillus rhamnosus. In alternative embodiments,probiotic comprise yeasts, e.g., comprise a Saccharomyces; or aSaccharomyces boulardii.

Probiotics, postbiotics, synbiotics etc. may at times be included assingle cultured components. They would avoid multiply culturedcomponents as they lose their implantation characteristics.

Preservatives, Cryoprotectants, Lyoprotectants

In alternative embodiments, to any composition as provided herein, forexample, a particle, a nanoparticle, a liposome, a tablet, a pill, acapsule, a gel, a geltab, a liquid, a powder, a suspension, a syrup, anemulsion, a lotion, an ointment, an aerosol, a spray, a lozenge, anophthalmic preparation, an aqueous or a sterile or an injectablesolution, a patch (optionally a transdermal patch or a medicatedadhesive patch), an implant, a dietary supplement, an ice cream, an ice,a yogurt, a cheese, an infant formula or infant dietary supplement, apasteurized milk or milk product or milk-comprising product, or a liquidpreparation embodiment or candies, lollies, drinks and the like, therecan be added various preservatives, cryoprotectants and/orlyoprotectants, including e.g., various polysaccharides or sugars (suchas sucrose, fructose, lactose, mannitol), glycerol, polyethylene glycol(PEG), trehalose, glycine, glucose, dextran and/or erythritol. Inalternative embodiments, other cryoprotectants that can be used areethylene glycol, 1,2-Propanediol, Methylcelliosolve, Dimethyl Formamide,or Dimethylsulphoxide Methanol. In alternative embodiments the contentof these cryoprotectants are between about 1% and about 50% butgenerally between about 5% and about 15% is adequate.

In alternative embodiments, a composition o as provided herein is frozenand/or is freeze-dried, or spray dried, or lyophilized, using any methodknown in the art. For example, a method for freeze-drying bacteriophagecan be used as described by Puapermpoonsiri et al., Int J. Pharm. 2010Apr. 15; 389(1-2):168-75, who used sucrose or poly(ethylene glycol) 6000to make bacteriophage-comprising freeze-dried cakes; or a method formaking freeze-dried formulations of bacteriophage encapsulated inbiodegradable microsphere, as described by Puapermpoonsiri et al.,European J. Pharmaceutics and Biopharmaceutics, Vol. 72, Issue 1, 2009,Pgs 26-33; or methods for making stable bacteriophage compositions ormatrices, as described e.g., by Murthy et al. WO2006047870 A1, or U.S.Pat. No. 8,309,077.

In alternative embodiments there are different types of final productsthat can be manufactured. In alternative embodiments a product orformulation as provided herein is a liquid. In alternative embodiments aproduct or formulation as provided herein is frozen and kept at e.g.minus 80 degrees for usage later given a cryoprotectant is added.

Biofilm Disrupting Compounds

In alternative embodiments, biofilm disrupting compounds added into acomposition or formulation o as provided herein (e.g., a product ofmanufacture, food, drink, nutraceutical, formulation, pharmaceutical orpharmaceutical preparation), or used to practice a method as providedherein. In alternative embodiments, in practicing the methods asprovided herein, biofilm disrupting compounds are administered before orduring (co-administered), or co-formulated with (e.g., in amultilaminated tablet or capsule), or separately formulated, as theadministered composition or formulation as provided herein. Inalternative embodiments, disrupting biofilms are used to separate fromthe colonic mucosa an adherent polysaccharide/DNA-containing layer, theso-called “biofilm”.

In alternative embodiments, other biofilm disrupting components oragents also can be used, e.g., enzymes such as a deoxyribonuclease(DNase), a N-acetylcysteine, an auranofin, alginate lyase, glycosidehydrolase dispersin B; Quorum-sensing inhibitors e.g., ribonucleic acidIII inhibiting peptide, Salvadora persica extracts,Competence-stimulating peptide, Patulin and penicillic acid;peptides—cathelicidin-derived peptides, small lytic peptide, PTP-7 (asmall lytic peptide, see e.g., Kharidia (2011) J. Microbiol.49(4):663-8, Epub 2011 Sep. 2), Nitric oxide, neo-emulsions; ozone,lytic bacteriophages, lactoferrin, xylitol hydrogel, synthetic ironchelators, cranberry components, curcumin, silver nanoparticles,Acetyl-11-keto-β-boswellic acid (AKBA), barley coffee components,probiotics, sinefungin, S-adenosylmethionine, S-adenosyl-homocysteine,Delisea furanones, N-sulfonyl homoserine lactones and/or macrolideantibiotics or any combination thereof.

In alternative embodiments, biofilm disrupting components or agents areadministered before and during the administration of a composition asprovided herein, e.g., as an antibacterial, in whatever format orformulation this may take place, for example, as a capsule.

In alternative embodiments, biofilm disrupting agents are added eitherbefore treatment and/or during and/or after treatment with a compositionas provided herein. In alternative embodiments, biofilm disruptingagents are used singly or in combination.

In alternative embodiments, biofilm disrupting agents include particularenzymes and degrading substances including in N-acetylcysteine,deoxyribonuclease (DNase). Others would include Alginate, lyase andGlycoside hydrolase dispersin, Ribonucleic-acid-III inhibiting peptide(RIP), Salvadora persica extracts, Competence-stimulating peptide (CSP)Patulin (PAT) and penicillic acid (PA)/EDTA, Cathelicidin-derivedpeptides, Small lytic peptide, PTP-7, Nitric oxide, Chlorhexidine,Povidone-iodine (PI), Nanoemulsions, Lytic bacteriophages,Lactoferrin/xylitol hydrogel, Synthetic iron chelators, Cranberrycomponents, Curcumin, Acetyl-11-keto-boswellic acid (AKBA), Barleycoffee (BC) components, silver nanoparticles, azithromycin,clarithromycin, gentamicin, streptomycin and also Disodium EDTA. Ozoneinsufflations of the colon can also be used to disrupt the biofilm.

Unit Dosage Forms and Formulations, Foods, and Delivery Vehicles

In alternative embodiments, a composition as provided herein (e.g., aparticle, a nanoparticle, a liposome, a tablet, a pill, a capsule, agel, a geltab, a liquid, a powder, a suspension, a syrup, an emulsion, alotion, an ointment, an aerosol, a spray, a lozenge, an ophthalmicpreparation, an aqueous or a sterile or an injectable solution, a patch(optionally a transdermal patch or a medicated adhesive patch), animplant, a dietary supplement, an ice cream, an ice, a yogurt, a cheese,an infant formula or infant dietary supplement, a pasteurized milk ormilk product or milk-comprising product) can be further processed by,e.g., spray-drying or equivalent, e.g., spray-drying in an inert gas orfreeze-drying under similar conditions, thus ending up with a powderedproduct.

In alternative embodiments, a composition o as provided herein can beformulated for enteral or parenteral administration, e.g., to reach thesystemic circulation, or for local delivery (e.g., for administration toskin, ears, teeth), as a topical for e.g., infections, as an inhalant,e.g., for inhalation of phages for the treatment of e.g., lunginfections, as described e.g., by Ryan et al. J Pharm Pharmacol. 2011October; 63(10):1253-64.

In alternative embodiments, a composition is manufactured, labelled orformulated as a liquid, a suspension, a spray, a gel, a geltab, asemisolid, a tablet, or sachet, a capsule, a lozenge, a chewable orsuckable unit dosage form, or any pharmaceutically acceptableformulation or preparation. In alternative embodiments, a composition asprovided herein is incorporated into a food or a drink (e.g., a yogurt,ice cream, smoothie), a candy, sweet or lolly, or a feed, a nutritionalor a food or feed supplement (e.g., liquid, semisolid or solid), and thelike.

For example, bacteriophage used to practice embodiments as providedherein can be encapsulated as described, e.g., by Murthy et al. in US2012-0258175 A1. A composition as provided herein can be manufactured,labelled or formulated as an orally disintegrating tablet as describede.g., in U.S. Pat. App. Publication No. 20100297031. A composition asprovided herein can be a polyol/thickened oil suspension as described inU.S. Pat. Nos. 6,979,674; 6,245,740. A composition as provided hereincan be encapsulated, e.g., encapsulated in a glassy matrix as describede.g., in U.S. Pat. App. Publication No. 20100289164; and U.S. Pat. No.7,799,341. A composition as provided herein can be manufactured,labelled or formulated as an excipient particle, e.g., comprising acellulosic material such as microcrystalline cellulose in intimateassociation with silicon dioxide, a disintegrant and a polyol, sugar ora polyol/sugar blend as described e.g., in U.S. Pat. App. PublicationNo. 20100285164. A composition as provided herein can be manufactured,labelled or formulated as an orally disintegrating tablet as describede.g., in U.S. Pat. App. Publication No. 20100278930. A composition asprovided herein can be manufactured, labelled or formulated as aspherical particle, as described e.g., in U.S. Pat. App. Publication No.20100247665, e.g., comprising a crystalline cellulose and/or powderedcellulose. A composition as provided herein can be manufactured,labelled or formulated as a rapidly disintegrating solid preparationuseful e.g. as an orally-disintegrating solid preparation, as describede.g., in U.S. Pat. App. Publication No. 20100233278. A composition asprovided herein can be manufactured, labelled or formulated as a solidpreparation for oral application comprising a gum tragacanth and apolyphosphoric acid or salt thereof, as described e.g., in U.S. Pat.App. Publication No. 20100226866.

A composition as provided herein can be manufactured, labelled orformulated using a water soluble polyhydroxy compound, hydroxycarboxylic acid and/or polyhydroxy carboxylic acid, as described e.g.,in U.S. Pat. App. Publication No. 20100222311. A composition as providedherein can be manufactured, labelled or formulated as a lozenge, or achewable and suckable tablet or other unit dosage form, as describede.g., in U.S. Pat. App. Publication No. 20100184785.

A composition as provided herein can be manufactured, labelled orformulated in the form of an agglomerate, as described e.g., in U.S.Pat. App. Publication No. 20100178349. A composition as provided hereincan be manufactured, labelled or formulated in the form of a gel orpaste, as described e.g., in U.S. Pat. App. Publication No. 20060275223.A composition as provided herein can be manufactured, labelled orformulated in the form of a soft capsule, as described e.g., in U.S.Pat. No. 7,846,475, or U.S. Pat. No. 7,763,276.

The polyols used in compositions as provided herein can be micronizedpolyols, e.g., micronized polyols, e.g., as described e.g., in U.S. Pat.App. Publication No. 20100255307, e.g., having a particle sizedistribution (dso) of from 20 to 60 μm, and a flowability below or equalto 5 s/100 g, or below 5 s/100 g.

In practicing embodiments as provided herein, a wide variation ofbacteriophage can be administered, for example, in some aspects, asmaller dosage can be administered because phage (i.e., bacteriophage)can replication in the host, i.e., in the individual to which acomposition as provided herein is administered. In alternativeembodiments, compositions as provided herein, including phage asprovided herein, are formulated per dose, or per serving, or per unitdosage at, or at a total daily dose of: between about 10(1) (or 10¹) and10(20) plaque-forming units (PFUs), or between about 10(3) and 10(17)PFUs, or between about 10(5) and 10(12) PFUs, or between about 10(7) and10(9) PFUs.

Gradual or Delayed Release Formulations

In alternative embodiments, provided are compositions formulated fordelayed or gradual enteric release comprising at least one active agent(e.g., a composition, a formulation or a pharmaceutical preparation asprovided herein) formulated with a delayed release composition orformulation, coating or encapsulation. In alternative embodiments,formulations or pharmaceutical preparations as provided herein aredesigned or formulated for delivery of active ingredient (e.g., abacteriophage) into the distal small bowel and/or the colon. Thus, forthis embodiment, it is important to allow the active ingredient to passthe areas of danger, e.g., stomach acid and pancreatic enzymes and bile,and reach undamaged to be viable in the distal small bowel andespecially the colon. In alternative embodiments, a formulation orpharmaceutical preparation as provided herein is a liquid formulation, amicrobiota-comprising formulation as provided herein and/or a frozen ora freeze-dried version thereof. In alternative embodiments, preferablyfor the encapsulated format, all are in powdered form.

In alternative embodiments, compositions as provided herein areformulated for delayed or gradual enteric release using celluloseacetate (CA) and polyethylene glycol (PEG), e.g., as described by Defanget al. (2005) Drug Develop. & Indust. Pharm. 31:677-685, who used CA andPEG with sodium carbonate in a wet granulation production process.

In alternative embodiments, compositions as provided herein areformulated for delayed or gradual enteric release using ahydroxypropylmethylcellulose (HPMC), a microcrystalline cellulose (MCC)and magnesium stearate, as described e.g., in Huang et al. (2004)European J. of Pharm. & Biopharm. 58: 607-614).

In alternative embodiments, compositions as provided herein areformulated for delayed or gradual enteric release using e.g., apoly(meth)acrylate, e.g. a methacrylic acid copolymer B, a methylmethacrylate and/or a methacrylic acid ester, a polyvinylpyrrolidone(PVP) or a PVP-K90 and a EUDRAGIT® RL PO™, as described e.g., in Kuksalet al. (2006) AAPS Pharm. 7(1), article 1, E1 to E9.

In alternative embodiments, compositions as provided herein areformulated for delayed or gradual enteric release as described in U.S.Pat. App. Pub. 20100239667. In alternative embodiments, the compositioncomprises a solid inner layer sandwiched between two outer layers. Thesolid inner layer can comprise a formulation or pharmaceuticalpreparation as provided herein and one or more disintegrants and/orexploding agents, one of more effervescent agents or a mixture. Eachouter layer can comprise a substantially water soluble and/orcrystalline polymer or a mixture of substantially water soluble and/orcrystalline polymers, e.g., a polyglycol. These can be adjusted in anexemplary composition as provided herein to achieve delivery of theliving components of an FMT distally down the bowel.

In alternative embodiments, compositions as provided herein areformulated for delayed or gradual enteric release as described in U.S.Pat. App. Pub. 20120183612, which describes stable pharmaceuticalformulations comprising active agents in a non-swellable diffusionmatrix. In alternative embodiments, a formulation or pharmaceuticalpreparation as provided herein is released from a matrix in a sustained,invariant and, if several active agents are present, independent mannerand the matrix is determined with respect to its substantial releasecharacteristics by ethylcellulose and at least one fatty alcohol todeliver bacteria distally.

In alternative embodiments, a formulation or pharmaceutical preparationas provided herein is formulated for delayed or gradual enteric releaseas described in U.S. Pat. No. 6,284,274, which describes a bilayertablet containing an active agent (e.g., an opiate analgesic), apolyalkylene oxide, a polyvinylpyrrolidone and a lubricant in the firstlayer and a second osmotic push layer containing polyethylene oxide orcarboxy-methylcellulose.

In alternative embodiments, a formulation or pharmaceutical preparationas provided herein is formulated for delayed or gradual enteric releaseas described in U.S. Pat. App. Pub. No. 20030092724, which describessustained release dosage forms in which a nonopioid analgesic and opioidanalgesic are combined in a sustained release layer and in an immediaterelease layer, sustained release formulations comprisingmicrocrystalline cellulose, EUDRAGIT RSPO™, CAB-O-SIL™, sodium laurylsulfate, povidone and magnesium stearate.

In alternative embodiments, a formulation or pharmaceutical preparationas provided herein is formulated for delayed or gradual enteric releaseas described in U.S. Pat. App. Pub. 20080299197, describing amulti-layered tablet for a triple combination release of active agentsto an environment of use, e.g., in the GI tract. In alternativeembodiments, a multi-layered tablet is used, and it can comprise twoexternal drug-containing layers in stacked arrangement with respect toand on opposite sides of an oral dosage form that provides a triplecombination release of at least one active agent. In one embodiment thedosage form is an osmotic device, or a gastro-resistant coated core, ora matrix tablet, or a hard capsule. In these alternative embodiments,the external layers may contain biofilm dissolving agents and internallayers the living bacteria.

In alternative embodiments, a formulation or pharmaceutical preparationas provided herein is formulated as multiple layer tablet forms, e.g.,where a first layer provides an immediate release of a formulation orpharmaceutical preparation as provided herein and a second layerprovides a controlled-release of another (or the same) formulation orpharmaceutical preparation as provided herein, or another active agent,as described e.g., in U.S. Pat. No. 6,514,531 (disclosing a coatedtrilayer immediate/prolonged release tablet), U.S. Pat. No. 6,087,386(disclosing a trilayer tablet), U.S. Pat. No. 5,213,807 (disclosing anoral trilayer tablet with a core comprising an active agent and anintermediate coating comprising a substantially impervious/impermeablematerial to the passage of the first active agent), and U.S. Pat. No.6,926,907 (disclosing a trilayer tablet that separates a first activeagent contained in a film coat from a core comprising acontrolled-release second active agent formulated using excipients whichcontrol the drug release, the film coat can be an enteric coatingconfigured to delay the release of the active agent until the dosageform reaches an environment where the pH is above four).

In alternative embodiments, a formulation or pharmaceutical preparationas provided herein is formulated for delayed or gradual enteric releaseas described in U.S. Pat. App. Pub. 20120064133, which describes arelease-retarding matrix material such as: an acrylic polymer, acellulose, a wax, a fatty acid, shellac, zein, hydrogenated vegetableoil, hydrogenated castor oil, polyvinylpyrrolidine, a vinyl acetatecopolymer, a vinyl alcohol copolymer, polyethylene oxide, an acrylicacid and methacrylic acid copolymer, a methyl methacrylate copolymer, anethoxyethyl methacrylate polymer, a cyanoethyl methacrylate polymer, anaminoalkyl methacrylate copolymer, a poly(acrylic acid), apoly(methacrylic acid), a methacrylic acid alkylamide copolymer, apoly(methyl methacrylate), a poly(methacrylic acid anhydride), a methylmethacrylate polymer, a polymethacrylate, a poly(methyl methacrylate)copolymer, a polyacrylamide, an aminoalkyl methacrylate copolymer, aglycidyl methacrylate copolymer, a methyl cellulose, an ethylcellulose,a carboxymethylcellulose, a hydroxypropylmethylcellulose, ahydroxymethyl cellulose, a hydroxyethyl cellulose, a hydroxypropylcellulose, a crosslinked sodium carboxymethylcellulose, a crosslinkedhydroxypropylcellulose, a natural wax, a synthetic wax, a fatty alcohol,a fatty acid, a fatty acid ester, a fatty acid glyceride, a hydrogenatedfat, a hydrocarbon wax, stearic acid, stearyl alcohol, beeswax,glycowax, castor wax, carnauba wax, a polylactic acid, polyglycolicacid, a co-polymer of lactic and glycolic acid, carboxymethyl starch,potassium methacrylate/divinylbenzene copolymer, crosslinkedpolyvinylpyrrolidone, polyvinylalcohols, polyvinylalcohol copolymers,polyethylene glycols, non-crosslinked polyvinylpyrrolidone,polyvinylacetates, polyvinylacetate copolymers or any combination. Inalternative embodiments, spherical pellets are prepared using anextrusion/spheronization technique, of which many are well known in thepharmaceutical art. The pellets can comprise one or more formulations orpharmaceutical preparations as provided herein, e.g., the liquidpreparation embodiment.

In alternative embodiments, a formulation or pharmaceutical preparationas provided herein is formulated for delayed or gradual enteric releaseas described in U.S. Pat. App. Pub. 20110218216, which describes anextended release pharmaceutical composition for oral administration, anduses a hydrophilic polymer, a hydrophobic material and a hydrophobicpolymer or a mixture thereof, with a microenvironment pH modifier. Thehydrophobic polymer can be ethylcellulose, cellulose acetate, cellulosepropionate, cellulose butyrate, methacrylic acid-acrylic acid copolymersor a mixture thereof. The hydrophilic polymer can bepolyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose,hydroxypropylmethyl cellulose, polyethylene oxide, acrylic acidcopolymers or a mixture thereof. The hydrophobic material can be ahydrogenated vegetable oil, hydrogenated castor oil, carnauba wax,candellia wax, beeswax, paraffin wax, stearic acid, glyceryl behenate,cetyl alcohol, cetostearyl alcohol or and a mixture thereof. Themicroenvironment pH modifier can be an inorganic acid, an amino acid, anorganic acid or a mixture thereof. Alternatively, the microenvironmentpH modifier can be lauric acid, myristic acid, acetic acid, benzoicacid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinicacid, adipic acid, sebacic acid, fumaric acid, maleic acid; glycolicacid, lactic acid, malic acid, tartaric acid, citric acid, sodiumdihydrogen citrate, gluconic acid, a salicylic acid, tosylic acid,mesylic acid or malic acid or a mixture thereof.

In alternative embodiments, a formulation or pharmaceutical preparationas provided herein is a powder that can be included into a tablet or asuppository. In alternative embodiments, a formulation or pharmaceuticalpreparation as provided herein can be a ‘powder for reconstitution’ as aliquid to be drunk or otherwise administered. In alternativeembodiments, a formulation or pharmaceutical preparation as providedherein is micro-encapsulated, formed into tablets and/or placed intocapsules, especially enteric-coated capsules.

Buffers and Antacids

In alternative embodiments, in practicing the methods as providedherein, buffers or antacids are administered before or during(co-administered), or co-formulated with a composition or formulation asprovided herein. For example, in alternative embodiments, a compositionor formulation as provided herein and a buffer or antacid areco-formulated, e.g., as multiple layer tablet form or as amulti-laminated tablet or capsule. In alternative embodiments of methodsas provided herein, buffers or antacids are separately formulated. Inalternative embodiments, the antacid, buffer or buffering agent isadministered (optionally before, during or after, or before and during,administration) to raise the pH of the stomach in the individual tobetween about 2.5 and 7, or between about 3 and 6.5, or to about 5.0,5.5, 6.0, 6.5, 6.8 or 7.0 (optionally these pH values reached before,during or after, or before and during, administration). In alternativeembodiments, the buffer or a buffering agent or the pharmaceuticallyacceptable excipient comprises an inorganic salt, a citric acid, asodium chloride, a potassium chloride, a sodium sulfate, a potassiumnitrate, a sodium phosphate monobasic, a sodium phosphate dibasic orcombinations thereof. In alternative embodiments, the antacid comprisesa calcium carbonate, a magnesium hydroxide, a magnesium oxide, amagnesium carbonate, an aluminum hydroxide, a sodium bicarbonate or adihydroxyaluminum sodium carbonate.

Infant Formulas, Feeds, Drinks, Candies, Nutritional or a Food or FeedSupplements

In alternative embodiments, a formulation or pharmaceutical preparationas provided herein is incorporated into an infant formula or equivalentinfant food or supplement, a food, a feed, a candy (e.g., a lollypop ora lozenge) a drink, a nutritional or a food or feed supplement (e.g.,liquid, semisolid or solid), and the like, as described e.g., in U.S.Pat. App. Publication No. 20100178413. In one embodiment, a formulationor pharmaceutical preparation as provided herein is incorporated into(manufactured as) a beverage as described e.g., in U.S. Pat. No.7,815,956. For example, a composition as provided herein is incorporatedinto a yogurt, an ice cream, a milk or milkshake, a “frosty”,“snow-cone”, or other ice-based mix, and the like.

In alternative embodiments, a formulation or pharmaceutical preparationas provided herein is a freeze-dried powder form added to a food, e.g.,a yogurt, an ice cream, a milk or milkshake, a “frosty”, “snow-cone”, orother ice-based mix, and the like. In one form of embodiments asprovided herein it can be kept in a lid-storage (e.g., of a yogurt orice cream) such that when it is twisted the powder falls into theproduct or formulation (e.g., yoghurt or ice cream) and then it can bestirred so as not to have the powder ferment ‘standing on the shelf’.Various flavourings can be added. In alternative embodiments, this isparticularly important for administration of a composition as providedherein, e.g., a wild type microbiota or a cultured bacteria, to a veryyoung individual and/or a patient with autism or related disease orcondition.

In alternative embodiments, these exemplary products are important whenadministered to children or babies who may have acquired variouspathogenic or abnormal bacteria, e.g., E. coli, Clostridia orDisulfovibrio, e.g., as in autism.

Packaging

In alternative embodiments, provided are compositions (e.g., a productof manufacture, food, drink, nutraceutical, formulation, pharmaceuticalor pharmaceutical preparation), including preparations, formulationsand/or kits, comprise combinations of ingredients, as described herein.In alternative embodiments, these combinations can be mixed andadministered together, or alternatively, they can be an individualmember of a packaged combination of ingredients, e.g., as manufacturedin a separate package, kit or container; or, where all or a subset ofthe combinations of ingredients are manufactured in a separate packageor container. In alternative aspects, the package, kit or containercomprises a blister package, a clamshell, a tray, a shrink wrap and thelike.

In one aspect, the package, kit or container comprises a “blisterpackage” (also called a blister pack, or bubble pack). In one aspect,the blister package is made up of two separate elements: a transparentplastic cavity shaped to the product and its blister board backing.These two elements are then joined together with a heat sealing processwhich allows the product to be hung or displayed. Exemplary types of“blister packages” include: Face seal blister packages, gang run blisterpackages, mock blister packages, interactive blister packages, slideblister packages.

Blister packs, clamshells or trays are forms of packaging used forgoods; thus, provided are blister packs, clamshells or trays comprisinga composition (e.g., a (the multi-ingredient combination of drugs asprovided herein) combination of active ingredients) as provided herein.Blister packs, clamshells or trays can be designed to be non-reclosable,so consumers can tell if a package has already opened. They are used topackage for sale goods where product tampering is a consideration, suchas the pharmaceuticals as provided herein. In one aspect, a blister packas provided herein comprises a moulded PVC base, with raised areas (the“blisters”) to contain the tablets, pills, etc. comprising thecombinations as provided herein, covered by a foil laminate. Tablets,pills, etc. are removed from the pack either by peeling the foil back orby pushing the blister to force the tablet to break the foil. In oneaspect, a specialized form of a blister pack is a strip pack. In oneaspect, in the United Kingdom, blister packs adhere to British Standard8404.

In one embodiment, provided is a method of packaging where thecompositions comprising combinations of ingredients as provided hereinare contained in-between a card and a clear PVC. The PVC can betransparent so the item (pill, tablet, geltab, etc.) can be seen andexamined easily; and in one aspect, can be vacuum-formed around a mouldso it can contain the item snugly and have room to be opened uponpurchase. In one aspect, the card is brightly colored and designeddepending on the item (pill, tablet, geltab, etc.) inside, and the PVCis affixed to the card using pre-formed tabs where the adhesive isplaced. The adhesive can be strong enough so that the pack may hang on apeg, but weak enough so that this way one can tear open the join andaccess the item. Sometimes with large items or multiple enclosed pills,tablets, geltabs, etc., the card has a perforated window for access. Inone aspect, more secure blister packs, e.g., for items such as pills,tablets, geltabs, etc. as provided herein are used, and they cancomprise of two vacuum-formed PVC sheets meshed together at the edges,with the informative card inside. These can be hard to open by hand, soa pair of scissors or a sharp knife may be required to open.

In one aspect, blister packaging comprises at least two or three or morecomponents (e.g., is a multi-ingredient combination o as providedherein: a thermoformed “blister” which houses multi-ingredientcombination as provided herein, and then a “blister card” that is aprinted card with an adhesive coating on the front surface. During theassembly process, the blister component, which is most commonly made outof PVC, is attached to the blister card using a blister machine. Thismachine introduces heat to the flange area of the blister whichactivates the glue on the card in that specific area and ultimatelysecures the PVG blister to the printed blister card. The thermoformedPVG blister and the printed blister card can be as small or as large asyou would like, but there are limitations and cost considerations ingoing to an oversized blister card. Conventional blister packs can alsobe sealed (e.g., using an AERGO 8 DUO™, SCA Consumer Packaging, Inc.,DeKalb Ill.) using regular heat seal tooling. This alternative aspect,using heat seal tooling, can seal common types of thermoformedpackaging.

Blister Packaging

In alternative embodiments, combinations of ingredients of compositionsas provided herein, or combinations of ingredients for practicingmethods as provided herein, can be packaged alone or in combinations,e.g., as “blister packages” or as a plurality of packettes, including aslidded blister packages, lidded blister or blister card or packets orpackettes, or a shrink wrap.

In alternative embodiments, laminated aluminium foil blister packs areused, e.g., for the preparation of drugs designed to dissolveimmediately in the mouth of a patient. This exemplary process compriseshaving the drug combinations as provided herein prepared as an aqueoussolution(s) which are dispensed (e.g., by measured dose) into analuminium (e.g., alufoil) laminated tray portion of a blister pack. Thistray is then freeze-dried to form tablets which take the shape of theblister pockets. The alufoil laminate of both the tray and lid fullyprotects any highly hygroscopic and/or sensitive individual doses. Inone aspect, the pack incorporates a child-proof peel open securitylaminate. In one aspect, the system give tablets an identification markby embossing a design into the alufoil pocket that is taken up by thetablets when they change from aqueous to solid state. In one aspect,individual ‘push-through’ blister packs/packettes are used, e.g., usinghard temper aluminium (e.g., alufoil) lidding material. In one aspect,hermetically-sealed high barrier aluminium (e.g., alufoil) laminates areused. In one aspect, any products of manufacture as provided herein,including kits or blister packs, use foil laminations and strip packs,stick packs, sachets and pouches, peelable and non-peelable laminationscombining foil, paper, and film for high barrier packaging.

In alternative embodiments, any multi-ingredient combinations orproducts of manufacture as provided herein, including kits or blisterpacks, include memory aids to help remind patients when and how to takethe drug. This safeguards the drug's efficacy by protecting each tablet,geltab or pill until it's taken; gives the product or kit portability,makes it easy to take a dose anytime or anywhere.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1: Exemplary Treatments

This example provides data demonstrating the efficacy of embodiments asprovided herein, and, while the invention is not limited by anyparticular mechanism of action, this example provides exemplarymechanisms by which exemplary embodiments provided antibacterialprotection to an individual in need thereof.

Here we show that phage-to-bacteria ratios were increased, relative tothe adjacent environment, on all mucosal surfaces sampled ranging fromcnidarians to humans. In vitro studies of tissue culture cells with andwithout surface mucus demonstrated that this mucus-dependent increase inphage abundance protects the underlying epithelium from bacterialinfection. Enrichment of phage in mucus occurs via interactions betweenmucin glycoproteins and immunoglobulin-like protein domains exposed onphage capsids. This interaction was found to be a result of phageimmunoglobulin-like domains binding variable glycan residues that coatthe mucin glycoprotein component of mucus. Metagenomic analysis foundthese immunoglobulin-like proteins present in many environments,particularly those adjacent to mucosal surfaces. Based on theseobservations, we present the Bacteriophage Adherence to Mucus (BAM)model, providing a ubiquitous but non-host-derived immune systemapplicable to mucosal surfaces. This model suggests that metazoanmucosal surfaces and phage have coevolved so that phage stick to them,thus benefiting the metazoan host through increased killing of mucosalbacteria, and the phage through increased interactions with bacterialhosts. The relationships shown here suggest a symbiotic relationshipbetween phage and metazoan hosts, and the data herein demonstrate anovel immunity for mucosal surfaces.

Here we demonstrate that phage adhere to mucus and that this associationreduces microbial colonization and pathology. In vitro studiesdemonstrated that this adherence was mediated by the interaction betweendisplayed Ig-like domains of phage capsid proteins and glycan residues,such as those in mucin glycoproteins. Homologs of these Ig-like domainsare encoded by phages from many environments, particularly thoseadjacent to mucosal surfaces. In alternative embodiments, provided arecompositions and methods that can incorporate the so-called“Bacteriophage Adherence to Mucus (BAM)” model, and comprise use ofphages to provide a non-host-derived antimicrobial defence on themucosal surfaces of diverse metazoan hosts.

Results

Phage Adhere to Mucus

Our preliminary investigations of mucosal surfaces suggested that phageconcentrations in the mucus layer were elevated compared to thesurrounding environment. Here we used epifluorescence microscopy tocount the phage and bacteria in mucus, sampled from a diverse range ofmucosal surfaces (e.g., sea anemones, fish, human gum), and in eachadjacent environment (FIG. S1). Comparing the calculatedphage-to-bacteria ratios (PBRs) showed that PBRs in metazoan-associatedmucus layers were on average 4.4-fold higher than those in therespective adjacent environment (FIG. 1A). The PBRs on these mucussurfaces ranged from 21:1 to 87:1 (average 39:1), as compared to 3:1 to20:1 for the surrounding milieus (average 9:1, n=9, t=4.719***P=0.0002). Earlier investigations of phage abundance in marineenvironments reported that phage typically outnumber bacteria by anorder of magnitude (49-51), but here we demonstrate that this margin wassignificantly larger in metazoan-associated mucus surface layers.

To determine if this enrichment was dependent on the presence of mucusrather than some general properties of the cell surface (e.g., charge),phage adherence was tested with tissue culture (TC) cells with andwithout surface mucus. In these assays, T4 phage were washed acrossconfluent cell monolayers for 30 min, after which non-adherent phagewere removed by repeated washings and the adherent phage quantified byepifluorescence microscopy. Two mucus-producing cell lines were used:T84 (human colon epithelial cells) and A549 (human lung epithelialcells). For these cells, mucin secretion was stimulated by pretreatmentwith phorbol 12-myristate-13-acetate (52, 53). Comparison of the T84cells with the non-mucus-producing Huh-7 human hepatocyte cell lineshowed that T4 phage adhered significantly more to the mucus-producingT84 cells (FIG. 1B, n>18, t=8.366, ****P<0.0001). To demonstrate themucus-dependence of this adherence, the mucus layer was chemicallyremoved from A549 cells by N-acetyl-L-cysteine (NAC) treatment (54)(FIG. S2). This significantly reduced the number of adherent phage tolevels similar to those observed with non-mucus-producing cell lines(FIG. 1B, n>40, t=9.561, ****P<0.0001). Lastly, we created an A549 shRNAmucus knockdown cell line (MUC⁻), reducing mucus production in A549, anda non-sense shRNA control (shConrol; FIGS. S3 and S4). Again, T4 phageadhered significantly more to the mucus-producing cells (FIG. 1B, n>37,t=7.673, ****P<0.0001).

While mucin glycoproteins are the predominant component of mucus, othermacromolecular components are also present, any of which might beinvolved in the observed phage adherence. We developed a modified topagar assay to determine whether phage adhered to a specificmacromolecular component of mucus. Plain agar plates and agar platescoated with 1% (w/v) mucin, DNA, or protein were prepared. Thatconcentration was chosen because it is at the low end of the range ofphysiological mucin concentrations (55). T4 phage suspensions wereincubated on the plates for 30 min after which the phage suspension wasdecanted to remove unbound particles. The plates were then overlaid witha top agar containing E. coli hosts and incubated overnight. The numberof adherent phage was calculated from the number of plaque forming units(PFU) observed. Significantly more T4 phage adhered to the 1%mucin-coated agar surface (FIG. 1c , n=12, t=5.306, ****P<0.0001).Combined, these three assays show that phage adhere to mucinglycoproteins.

Phage Adherence and Bacterial Infection

The mucus layer is an optimal environment for microbial growth,providing structure and access to a range of diverse mucin-associatedglycans. To limit this growth the metazoan host retards microbialcolonization by diverse antimicrobial mechanisms (23, 25-27). Thisraises the question of whether the increased number of adherent phagefound on mucosal surfaces, provides the metazoan host with the benefitof reduced microbial colonization? To test this prediction, bacterialattachment to mucus-producing and non-mucus-producing TC cells wasassayed both with and without pretreatment with the mucus-adherent phageT4. Here confluent monolayers of TC cells were overlaid with T4 phagefor 30 min, washed to remove non-adherent phage, and then incubated withE. coli for 4 h. Cells were then scraped from the plates and attachedbacteria were fluorescently stained and counted by epifluorescencemicroscopy. Phage pretreatment of mucus-producing TC cell lines (T84,A549) significantly decreased subsequent bacterial attachment (FIG. 2a ,T84; n>30, t=32.05, ****P<0.0001, A549; n>30, t=36.85, ****P<0.0001).Comparatively, phage pretreatment of non-mucus producing cells (Huh-7;MUC, an A549 mucus knockdown strain), resulted in less dramatic shiftsin bacterial attachment. These results show that pretreatment of amucosal surface with phage reduces adherence of a bacterial pathogen.

To test whether the observed reduced bacterial adherence was accompaniedby reduced pathology of the underlying TC cells, mucus-producing A549and non-mucus-producing MUC TC cells were exposed to E. coli overnight,either with or without a 30 min pre-treatment with T4 phage. Infectionwas quantified as the percentage of cell death. Adherence of phageeffectively protected the mucus-producing cells against the subsequentbacterial challenge (FIG. 2b , n=12, ****P<0.0001). Phage pretreatmentshowed a reduced protection to the non-mucus-producing MUC⁻ cellsagainst bacterial challenge, decreasing cell death by only 2-fold.Notably, phage pretreatment of mucus-producing A549 cells, relative tothe non-mucus-producing MUC⁻ cells, resulted in a 3.6-fold greaterreduction in cell death (n=12, *P=0.0181). Results show that mucinproduction significantly increased phage-associated protection of tissueculture cells, compared to mucin knockdown cell lines.

Role of Capsid Ig-Like Domains in Phage Adherence

Minot et al. recently reported that human gut-associated phagecommunities encode a diverse array of hypervariable proteins, includingsome with hypervariable Ig-like domains (43). T4 phage contain fourIg-like domains as part of the structural protein, Hoc, 155 copies ofwhich are displayed on the phage capsid surface (56, 57). Based on this,and given that most Ig-like domains function in recognition and adhesionprocesses, we hypothesized that the T4 Hoc protein might mediate theadherence of T4 phage to mucus. To test this, the adherence of hoc⁺ T4phage and a hoc⁻ mutant to mucin-, DNA-, and protein-coated agar plateswas compared to an agar control using the modified top agar assay (seeabove). Relative to plain agar, the adherence of hoc⁺ T4 phage increased4.1-fold for mucin-coated agar (n>11, t=3.977, ***P=0.0007), whereasadherence increased only slightly for agar coated with DNA (1.1-fold) orprotein (1.2-fold; FIG. 3a ). Unlike the hoc⁺ T4 phage, the hoc⁻ phagedid not show preferential adherence to the mucin-coated agar, withincreased adherence of 1.2-, 1.2-, and 1.1-fold for mucin, DNA, andprotein coatings, respectively. To ensure that none of themacromolecules directly affected phage infectivity, hoc⁺ and hoc⁻ T4phage were incubated 1% (w/v) solutions of mucin, DNA, or BSA. Phagesuspensions were combined with E. coli top agar as described above andlayered over uncoated agar plates. The results confirmed that themacromolecules did not affect phage infectivity (FIG. S5). To furtherdemonstrate the mucin adherence capability of other phage capsiddisplayed Ig-like domains, we repeated the modified top agar assay usingIg⁺ and Ig T3 phage. Results indicate a similar increase in adherence tomucin for the Ig⁺ T3 phage, while Ig T3 phage did not show this increase(FIG. S6).

Next a competition assay using hoc⁺ and hoc⁻ T4 phage and mucusproducing TC cells was performed. Phage suspended in mucin solutionsranging from 0% to 5% (w/v), were washed over confluent layers ofmucus-producing A549 TC cells and phage adherence assayed as describedabove. Adherence of hoc⁺ T4 phage, but not of hoc⁻ T4 phage, was reducedby mucin competition in a concentration-dependent manner (FIG. 3b ).

Interaction of the Hoc protein domains displayed on the capsid surfacewith mucin glycoproteins could potentially affect the rate of diffusionof T4 virions in mucus. To evaluate this, we used multiple-particletracking (MPT) to quantify transport rates of phage particles in bufferand in mucin suspensions. The ensemble average effective diffusivity(D_(eff)) calculated at a time scale of 1 s for both hoc⁺ and hoc⁻ T4phage in buffer was compared against that in 1% (w/v) mucin suspensions.Both hoc⁺ and hoc⁻ phage diffuse rapidly through buffer (FIG. 3c ).While hoc⁻ phage diffused in 1% mucin at the same rate as in buffer, themucin decreased the diffusion rate for hoc⁺ phage particles 8-fold.Combined these three experiments support the hypothesis that the Hocproteins displayed on the T4 phage capsid interact with mucin, therebyslowing phage diffusion in mucus and increasing phage mucosal residencetime.

Phage Capsid Ig-Like Domains Interact with Glycans

It is known that approximately 25% of sequenced tailed dsDNA phages(Caudovirales) encode structural proteins with predicted Ig-like domains(47). A search of publicly available viral metagenomes for homologs ofthe Ig-like domains of the T4 Hoc protein yielded numerous viral Ig-likedomains from a variety of environments (FIG. 4a ). These domains weremore likely to be found in samples collected directly from mucus (e.g.,sputum samples) or from an environment directly adjacent to a mucosalsurface (e.g., intestinal lumen, oral cavity). All homologs displayedhigh structural homology (Phyre2 confidence score average 96±5%) with aplant-sugar binding domain known for its promiscuous carbohydratebinding specificity (Supplementary Methods and Table S1), suggesting aninteraction between these Ig-like domains and glycans.

Mucins are complex glycoproteins with highly variable glycan groupsexposed to the environment. To investigate whether Hoc interacts withglycans, and if so, to determine whether it interacts with a specificglycan or with a diverse array of glycans, we assayed phage adherence tomicroarrays printed with 610 mammalian glycans. While hoc⁺ T4 phageadhered to a large number of diverse glycans, they showed a preferencefor the O-linked glycan residues that are typically found in mucinglycoproteins (FIG. 4b ; Table S2). The hoc⁻ T4 phage exhibitedsignificantly lower affinity for all tested glycans. This indicates thatHoc mediates interactions between T4 phage and varied glycan residues.

Discussion

In diverse metazoans, body surfaces that interact with the environmentare covered by a protective layer of mucus. These mucus layers providefavorable habitats for bacteria that serve as the point of entry formany pathogens but also support large populations of microbialsymbionts. Also present are diverse phages that prey on specificbacterial hosts. Moreover, phage concentrations in mucus are elevatedrelative to the surrounding environment (an average 4.4-fold increasefor a diverse sample of invertebrate and vertebrate metazoans; FIG. 1A).The increased concentration of lytic phage on mucosal surfaces providesa previously unrecognized metazoan immune defence effected by phagelysis of incoming bacteria.

Working with a model system employing T4 phage and various tissueculture (TC) cell lines, we demonstrated that the increasedconcentration of phage on mucosal surfaces is mediated by weak bindinginteractions between the variable Ig-like domains on the T4 phage capsidand mucin-displayed glycans. The immunoglobulin protein fold is wellknown for its varied but essential roles in the vertebrate immuneresponse and cell adhesion. Ig-like domains are also present inapproximately one quarter of the sequenced genomes of tailed DNA phages(47). Notably, these domains were found only in virion structuralproteins and are typically displayed on the virion surface. Thus theywere postulated to bind to bacterial surface carbohydrates duringinfection (47, 48). However, mucin glycoproteins, the predominantmacromolecular constituent of mucus, display hundreds of variable glycanchains to the environment that offer potential sites for binding byphage Ig-like proteins. Furthermore, we speculate that phage utilize thevariability of the Ig-like protein scaffold (supporting >10¹³ potentialalternatives) to adapt to the host's ever-changing patterns of mucinglycosylation.

The presence of an Ig-like protein (Hoc) displayed on the capsid of T4phage significantly slowed the diffusion of the phage in mucinsolutions. In vivo, similar phage binding to mucin glycans wouldincrease phage residence time in mucus layers. Since bacterialconcentrations are typically enriched in mucus, we predict thatmucus-adherent phage are more likely to encounter bacteria, potentiallyincreasing their replicative success. If so, phage Ig-like domains thatbind effectively to the mucus layer would be under positive selection.Likely Hoc and other phage proteins with Ig-like domains interact withother glycans with different ramifications, as well (48, 57).

Previous metagenomic studies had documented the ubiquity and diversityof bacteria and phage within mucus-associated environments (e.g., humangut, human respiratory tract, corals) (51, 58-63). We had also becomeaware of some of the essential but adaptable services provided bysymbiotic bacteria in these environments (McFall-Ngai et al., 2013).However, only recently have we begun to investigate the dynamicinfluences of phage within host-associated ecosystems (36, 43, 64). Inthis work, we used an in vitro model system to demonstrate a mechanismof phage adherence to the mucus layers that shield metazoan cells fromthe environmental. Further, adherent phage protected the underlyingepithelial cells from bacterial infection. Based on these observationsand previous research, we describe the Bacteriophage Adherence to Mucus(BAM) model of immunity in which the adherence of phage to mucosalsurfaces yields a non-host-derived, antimicrobial defence. According tothis model (summarized in FIG. 5), the mucus layer, already consideredto be part of the innate immune system and known to provide physical andbiochemical anti-microbial defences (15, 23, 65), also accumulatesphage.

The model system we employed involved a single lytic phage and hostbacterium. Within the mucosal layer reside diverse bacterial lineagesand predictably an even greater diversity of phage strains enmeshedwithin complex phage-bacterial infection networks and engaged in adynamic arms race (66, 67). These and other factors lower theprobability that any given phage-bacterium encounter will result in asuccessful infection. The time dimension adds further complexity. Themucus layer is dynamic. Mucins are continually secreted by theunderlying epithelium; mucus is continually sloughed from the outersurface. As a result, there is an ongoing turnover of both the bacterialand phage populations in the mucus layer. Driven by kill-the-winnerdynamics, the population of phage types that can infect the dominantbacterial types present will cycle along with the populations of theirhosts. Through such mechanisms we envision that adherent lytic phagesprovide a dynamic and adaptable defence for their metazoan hosts—thefirst example of a metazoan-phage symbiosis.

In summary, compositions and methods as provided herein, which canincorporate “BAM immunity”, reduce bacterial pathogenesis and provide apreviously unrecognized, mucosal immunity.

Materials and Methods

Bacterial and Phage Counts from Mucus and Environmental Samples

Samples of mucus and the adjacent environment were collected directlyfrom nine evolutionarily-diverse mucosal surfaces (FIG. S1 and TableS1). Samples were transported and maintained on ice until processed. Allsamples were fixed overnight in 0.5% glutaraldehyde at 4° C., thenincubated in 6.5 mM dithiothreitol (DTT) at 37° C. for one hour toassist mucus degradation. A 1-100 μl aliquot was diluted with 2 ml of0.02 μm SM buffer, briefly mixed, then filtered onto a 0.02 μm anodiscpolycarbonate filter (Whatman, Piscataway, N.J.). Filters were stainedwith 10×SYBR GOLD™, washed, and visualized on a ZEISS™ epifluorescencemicroscope. For each sample, 20-30 images were taken for both bacteriaand virus-like particles. Images were analyzed using IMAGE PRO PLUS 5.1™software (MediaCybernetics, Rockville, Md.). Counts of bacteria andvirus-like particles (referred to as “phage” throughout the text) per mlwere made as previously described (68).

Phage Adherence to Mucus-Associated Macromolecules

LB agar plates were coated with 1 ml of 1% (w/v) of one of the followingin 1×PBS: type III porcine stomach mucin, DNA from salmon testes, orbovine serum albumin (all three from Sigma-Aldrich, St. Louis, Mo.) andthen allowed to dry. Stocks of hoc⁺ and hoc⁻ T4 phage (10⁹ PFU ml⁻¹)were serially diluted to 1×10⁷ and 1×10⁻⁸ per ml in LB, and a 5 mlaliquot of each dilution was washed across the plates for 30 min at 37°C. on an orbital shaker. After the phage suspensions were decanted fromthe plates, the plates were shaken twice to remove excess liquid anddried. Each plate was then layered with 1 ml of overnight E. coliculture (10⁹ ml⁻¹) in 3 ml of molten top agar and incubated overnight at37° C. The number of adherent phage was calculated from the number ofplaque forming units (PFU) observed multiplied by the initial phagedilution. To determine if mucus macromolecules directly affected phageinfectivity, hoc⁺ and hoc⁻ T4 phage (10⁹ PFU ml⁻¹) were serially dilutedas described above into 1 ml LB solutions containing 1% (w/v) mucin,DNA, or BSA. After incubation for 30 min at 37° C., the phagesuspensions were combined with E. coli top agar as described above andlayered over uncoated agar plates (FIG. S6).

Tissue Culture Cells and Mucus Reduction

Monolayers of various mucus-producing and non-mucus-producing tissueculture (TC) cells (Supplementary Methods) were grown to confluence in 6well multi-well Tissue Culture plates (Becton Dickinson, Franklin Lakes,N.J.). (a) Mucus-producing TC cells were exposed to 1 μg/ml of a phorbolester, phorbol-12-myristate-13-acetate (PMA: Sigma-Aldrich, St. Louis,Mo.) in the culture media overnight to stimulate the mucin secretoryresponse (53). (b) The mucolytic agent N-acetyl-L-cysteine (NAC;Sigma-Aldrich, St. Louis, Mo.) was used to chemically remove mucus fromA549 TC cells (60 mM NAC in serum-free media for 1 h with agitation)(54). Mucus depletion was confirmed using PAS/AB (FIG. S2). (c) A MUC⁻A549 cell line was produced by transduction of A549 cells with GIPZLentiviral Human MUC1 shRNA and TRIPZ Inducible Lentiviral Human MUC5ACshRNA as target vectors; a shControl A549 cell line was produced usingthe GIPZ Non-silencing Lentiviral shRNA Control as a control vector(Thermo Scientific, Waltham, Mass.; Supplementary Methods). Knockdown ofmucus production in the MUC⁻ cell line was confirmed by Western blotanalysis and periodic acid-Schiff-Alcian blue (PAS/AB; Sigma-Aldrich,St. Louis, Mo.; FIGS. S3 and S4).

Phage Treatment of TC Cells

TC cells were washed twice with 5 ml of serum-free media to removeresidual antibiotics, layered with 2 ml of serum-free media containingT4 phage (10⁷ or 10⁹ ml⁻¹), and incubated at 37° C. and 5% CO₂ for 30min. Cells were then washed five times with 5 ml of serum-free media toremove non-adherent phage.

Phage Adherence to TC Cells

TC cells were treated with phage (10⁹ ml⁻¹; see above), then scrapedfrom plates using CORNING CELL SCRAPERS™ (Sigma-Aldrich, St. Louis,Mo.). Adherent phage were counted by epifluorescence microscopy asdescribed above.

Bacterial Adherence to TC Cells with/without Phage Pretreatment

TC cells, with or without pretreatment with T4 phage (10⁷ ml⁻¹) werelayered with 2 ml serum-free media containing E. coli (10⁷ ml⁻¹),incubated at 37° C. and 5% CO₂ for 4 h, and then washed five times with5 ml serum-free media to remove non-adherent phage and bacteria. Cellswere scraped from plates and adherent phage and bacteria were counted byepifluorescence microscopy as described above.

TC Cell Death from Bacterial Infection

Mucus-producing A549 and MUC⁻ A549 TC cells were grown to confluence. T4phage were cleaned using Amicon 50™ kDa centrifugal filters (Millipore,Billerica, Mass.) and SM buffer to remove bacterial lysis products.Cells, with or without T4 phage pretreatment (10⁷ ml⁻¹), were incubatedwith E. coli (10⁷ ml⁻¹) overnight. Afterwards, TC cells were recoveredfrom the plates by trypsin/EDTA solution (Invitrogen, Grand Island,N.Y.). Cells were pelleted by centrifugation and resuspended in 1×PBS.Dead cells were identified by staining with 1 mg/ml of propidium iodide(Invitrogen, Grand Island, N.Y.). Samples were then analyzed on aFACSCANTO II™ flow cytometer (BD Biosciences, San Jose, Calif.) withexcitation at 488 nm and emission detected through a 670 LP filter. Theforward scatter threshold (FSC) was set at 5,000 and a total of 10,000events were collected for each sample.

Mucin Competition Assay

Mucus-producing A549 TC cells were grown to confluence. Hoc⁺ and hoc⁺ T4phage (10⁹ ml⁻¹) were diluted into mucin solutions ranging between 0%and 5% (w/v), then washed over TC cells for 30 min at 37° C. and 5% CO₂.Cells were washed five times with 5 ml serum-free media to removenon-adherent phage, scraped from plates, and adherent phage werequantified as described above.

Multiple Particle Tracking (MPT)

Assays were performed in plastic well chambers mounted on glass slidesthat had been coated with poly(dimethylsiloxane) (PDMS) to prevent phageadherence. 5 μl of 10⁹ ml⁻¹ SYBR™ Gold-labelled phage suspensions wereadded to 50 μl of 1% (w/v) mucin solution in 1×PBS buffer. Trajectoriesof fluorescently labelled phage were observed using a DELTAVISIONSPECTRIS™ Model DV4™ deconvolution microscope (Applied Precision,Issaquah, Wash.) equipped with a 100× Olympus PLANAPO 1.4™ lens. Movieswere captured using SOFTWORX® 5.0.0 (Applied Precision, Issaquah,Wash.); 100 ms temporal resolution for 30 s; ten analyses per sample;n>100 particle trajectories per analysis. Trajectories were analyzedwith the SPOTTRACKER™ 2D and 3D plugin for Image J (69). The coordinatesof phage particle centroids were transformed into time-averaged meansquare displacements (MSD): <Δr²(τ)>=<Δx²+Δy²>, from which effectivediffusivities (<D_(eff)>) were calculated; D_(eff)=<Δr²(τ)>/(4τ) (70,71).

Glycan Microarray

Phage binding to glycans was assayed using printed mammalian glycanmicroarrays (version 5.1, Consortium for Functional Glycomics Core)containing 610 glycan targets. Samples of hoc⁺ T4 phage, hoc⁻ T4 phage,and buffer controls were applied to separate glycan microarray slides.Each slide received 35 μl of sample, 35 μl of binding buffer (Trissaline with 2 mM Ca²⁺, 2 mM Mg²⁺, 1% BASE and 0.05% Tween 20), and acoverslip. Slides were incubated for 1 h at room temperature and washedwith binding buffer. Slides were then incubated in SYBR GOLD™fluorescence dye (diluted 1:10,000 in binding buffer) for 1 h under acoverslip at room temperature, washed, dried, and immediately scanned ina PerkinElmer PROSCANARRAY MICROARRAY SCANNER™ using an excitationwavelength of 488 nm. IMAGENE™ software (BioDiscovery, Inc., El Segundo,Calif.) was used to quantify fluorescence. Normalized relativefluorescence (RFU) values reported are the average (after subtraction ofbackground buffer fluorescence) from six spots for each glycanrepresented on the array.

Graphing and Statistics:

Graphing and statistical analyses were performed using GRAPHPAD PRISM 6™(GraphPad Software, San Diego, Calif.). All error bars represent 5-95%confidence intervals. The mid-line represents the median and the meanfor box plots and bar plots, respectively.

Example 2: Exemplary Treatments

This example provides data demonstrating the efficacy of exemplaryembodiments as provided herein, and, while the invention is not limitedby any particular mechanism of action, this example provides exemplarymechanisms by which aspects as provided herein provide antibacterialprotection to an individual in need thereof.

Here we tested a phage therapy application of the bacteriophageadherence to mucus (BAM) model on a life-like mucosal surface. Onlymucus-adherent phage were capable of reducing bacterial colonization ofthe mucus layer, with non-adherent phages performing no better than whenno phage were added. Diffusion experiments reveal that mucus-adherentphage exhibit continuous time random walk (CTRW) subdiffusive motion inmucus solutions, which theoretically predicts increased phage-bacteriaencounters. Using the classical adsorption assay, we experimentallyvalidated that the subdiffusive motion of phage is a more effectivesearch strategy bacterial hosts within mucus. Here we demonstrate thatphage are capable of mimicking complex search patterns that lead toincreased encounter rates, effectively enabling phage to hunt theirbacterial prey within mucosal surfaces. The evidence shown here proposesphage as the first predator known to utilize a subdiffusive searchstrategy, and, while the invention is not limited by any particularmechanism of action, further demonstrates the success of mucosal phagetherapies provided herein.

In an attempt to understand the fundamental interactions occurringbetween phage and bacterial host within the context of the mucus layer,we developed a microfluidic system to simulate a life-like mucosalsurface and test the phage therapy application of the BAM model (Barr,Auro, et al., 2013). Here we used the T4 phage model system, includingthe mucus-adherent T4 wild type phage (T4 wt), and thenon-mucus-adherent, T4 Hoc knockout phage (T4 hoc⁻)—a deletion mutantthat does not contain the Hoc domain but is otherwise a normallyinfective T4 phage particle. Microfluidic chips allowed for theinvestigation of well-controlled phage and bacterial infections on arepeatable and standardized mucosal surface, with constant fluid-flowselectively amplifying microbial growth within the mucus layer.

We investigated how both mucus-adherent and non-adherent phages diffusewithin simplified mucin solutions. Using the classical phage adsorptionassay, we then tested two models of diffusion within relevant mucosalenvironments, and propose a potential therapeutic solution for theinconsistencies associated with mucosal phage therapy applications.

Phage Therapy within a Dynamic Mucosal Surface

A mucosal microfluidic device (chip) was designed to mimic themicroenvironment of a mucosal surface, exhibiting constant fluid flow,physiological shear stress, and mucus secretion dynamics (McGuckin <i>etal.</i>, 2011). The chip was made of poly(dimethylsiloxane) (PDMS) andconsisted of a single microfluidic channel, with in and out ports,attached to a glass microscope slide (FIGS. 1A & B, Example 2; or FIGS.23A, 23B). Mucus-producing human lung epithelial cells were seeded intothe channel, and allowed to attach to the glass surface. Tissue culturemedia was then perfused through at a flow rate of 40-100 μL h⁻¹ (0.02dyne cm⁻²) to mimic the physiological fluid flow and shear stressesexperienced by a mucosal surface (Kim et al., 2012; Ishikawa et al.,2011). Using a multiplex syringe-pump, up to nine of these chips weresimultaneously fed with continual fluid-flow for seven days to allow theestablishment of a confluent mucosal epithelium exhibiting mucussecretion and turnover dynamics (FIGS. 1C, D & E, Example 2; or FIGS.23C, 23D and 23E). Using these chips we performed perfusion-based, phageand bacterial infection assays upon the in vitro mucosal surface. Morespecifically, the continually fluid-flow allowed us to selectivelyamplify the effects of microbial growth within the mucosal surface, asboth contaminating growth from the surrounding milieu and sloughed mucuslayers were constantly washed away and removed from the system.

FIG. 1, Example 2; or FIG. 23, schematically and graphically illustratesmicrofluidic devices (chip) designed to simulate a life-like in vitromucosal surface, and their function, respectively. A) Schematic of chipdesign and measurements. B) Single chip bonded to a glass microscopeslide with microfluidic tubing attached to in and out ports. C)Mucus-producing lung tissue cultured cells seeded into main channel. D)Cells in main after seven days of fluid flow and growth. E) Multiplexsyringe pump running nine chips simultaneously. F) Phage therapy assaywith phage and bacterial counts taken from the mucosal surface. G) Phagedetachment rates from mucosal surface over a six hour period, dashedline indicates the 1 hr wash time point used in phage therapy assay.

One critical aspect of phage therapy that has never been effectivelyinvestigated in vitro is the treatment of such a dynamic and life-likemucosal surface. To investigate the tripartite interactions betweenphage-bacterium-mucosa, we treated chips with either the mucus-adherentT4 wt phage (10⁷ mL⁻¹), the non-adherent T4 hoc⁻ phage (10⁷ mL⁻¹), or ano-phage control, for 24 hours in order to saturate the mucosalepithelium. Chips were then washed for one hour to remove phage notenmeshed within the mucus layer, followed by infection with Escherichiacoli (E. coli) bacterial host (10⁷ mL⁻¹) at a multiplicity of infection(MOI) of 1. Following infection, chips were washed with phage- andbacterium-free media for 18 hours, allowing for microbial replicationwithin the mucus layer. Chips were then sacked, the mucosal surfacescraped, and the number of phage and bacteria present within the mucuslayer was quantified by culturing (FIG. 1F, Example 2, or FIG. 23F).High phage abundances were detected in both the T4 wt and hoc⁻treatments, indicating that both phage populations were activelyinfecting and propagating with the E. coli bacterial host. Yet only theT4 wt treated chips showed a significant antimicrobial effect, with overa 4000-fold reduction in bacterial abundance within the mucus layer(n=5, t=; P<Barb stats to come). Comparatively, the T4 hoc⁻ treatedchips showed no effective reduction in bacterial mucosal abundancecompared to the no-phage control (n=5, t=; P=).

Why were only the mucus-adherent, T4 wt phage effective at reducing thebacterial load on the in vitro mucosal surface? The mucus layer is acomplex, gel-like, network that traps and embeds both T4 wt and hoc⁻phage particles within it. Thus in an attempt to explain the differencesin anti-bacterial protection, we investigated whether mucus-adherent T4wt phage accumulated at higher abundance, or persisted for longer,within the chips mucosal surface, than the non-adherent T4 hoc⁻ phage(Barr, Auro, et al., 2013). Chips were again saturated with either T4 wtor hoc⁻ phage (10⁷ mL⁻¹) for 24 hours, and then washed with phage- andbacterium-free media to quantify phage detachment from the mucosalsurface over a six-hour period (FIG. 1G, Example 2, or FIG. 23G).Detachment of T4 wt and hoc⁻ phage from the mucosal surface within thefirst hour were comparable, with the majority of both phages rapidlyreleasing from the mucus layer. At this point, a large portion of phagehad detached from the mucosal surface, and the one-hour wash from theprevious infection assay likely resulted in a MOI of significantly lessthan 1. The following five hours showed a much slower rate of detachmentfor both phage types, with slightly higher concentrations of T4 wt phagedetaching from the mucosal surface. Results suggest that neithermucus-adherent phage accumulation, nor persistence within the mucosalsurface were responsible for differences in antimicrobial protection.Thus the weak BAM mechanisms likely do not govern phage persistencewithin mucosal surface, and instead mucus secretion and turnoverdynamics regulate mucosal phage abundances.

Bacteriophage Exhibit Subdiffusive Motion in Mucus.

In an attempt to elucidate the above-mentioned differences between T4 wtand hoc⁻ phage within our mucosal surface, we employed high-speedmultiple particle tracking (MPT) to track the diffusion of both phagetypes across a physiological range of homogenous mucin solutions.Fluorescence-labelled phage were mixed with 0% (buffer), 0.2%, 1%, and5% mucin solutions (wt/vol) within a microscope slide well, and trackedat a temporal resolution of 43.5 ms. We manually tracked the diffusionof phage particles and calculated their diffusion constants (μm²/s)(FIG.2A, Example 2, or FIG. 24A) (Meijering et al., 2012).

Diffusion of both phage were comparable in 0% solutions, but T4 wt phagediffused 1.13 and 1.62 times slower than the T4 hoc⁻ phage, in 0.2% and1% mucin solutions respectively. These 1% mucin diffusion values aredifferent from our previously reported results (Barr, Auro, et al.,2013), and are likely due to higher temporal resolution and improve MPTmethodologies employed here. Interestingly at 5% mucin concentrationsboth T4 wt and hoc⁻ phage particles were effectively ‘trapped’ in themucin solution, with diffusion constants approaching our resolvingpower.

It is known that within complex fluids, such as the cellular cytoplasmor mucus gels, that diffusion can exhibit anomalous characteristics overtime that are not defined by normal Brownian motion (Guigas & Weiss,2008; Saxton, 2007). Enhancing diffusion results in superdiffusion,while subdiffusion is a result of hindrance or slowing. Normal diffusionis characterized by a MSD that grows linearly over time (τ) with thediffusion constant (D); that is, MSD(τ)=Dr. Comparatively, anomalousdiffusion is characterized by the power-law exponent alpha (a), whereMSD˜τ^(α). Simply put, normal diffusion is characterized by α=1,superdiffusion by α>1, and subdiffusion by α<1. Mucus-adherent phagebinding to glycan residues displayed on the large mucin glycoproteins,slowing their diffusion through the mucus network, but this slowing maynot be anomalous over time. To investigate whether T4 wt and hoc⁻ phageexhibit anomalous diffusion in mucus, we calculated their α-value offrom our MPT experiments (FIG. 2B, Example 2, or FIG. 24B). Allexperimental conditions revealed normal diffusion (i.e., α≈1), with theexception of T4 wt phage in 0.2% and 1% mucin solutions, both showing aclear subdiffusive signal of α=0.94 and α=0.81, respectively.

FIG. 2, Example 2, or FIG. 24, graphically illustrates: A)Mucus-adherent T4 wt and non-adherent T4 hoc⁻ phage diffusion constants(μm²/s) in 0% (buffer), 0.2%, 1%, and 5% mucin solutions (wt/vol). B)Alpha value (α) measurements of T4 wt and T4 hoc⁻ phage in 0% (buffer),0.2%, 1%, and 5% mucin solutions (wt/vol). Brownian diffusion α≈1,subdiffusion α<1.

Subdiffusion has been found to emerge from a wide variety of complexprocesses, including the intracellular transport of proteins, nucleicacids, and lipids. But the picture is not simple, with different modelsof subdiffusion having distinct biophysical effects. Next we calculatedthe ensemble-averaged MSD for all experimental conditions, with alphavalues represented by the slope of the line of best fit showing adecreased slope for T4 wt phage in 0.2% and 1% mucin solutions (FIG. 3,Example 2, or FIG. 25). To elucidate the most appropriate model ofsub-diffusion, we compared these results with a time-averaged MSDcalculation, which revealed Brownian motion for both phages in allexperimental conditions (α≈1). This ensemble-averaged aging effect, orweak ergodicity breaking, is the hallmark feature described by acontinuous time random walk (CTRW) model (Ernst et al., 2014). Accordingto the CTRW model of sub-diffusion, particles diffuse by Brownianmotion, but are interrupted by periods of the particle becoming ‘stuck’for periods of ever increasing time, which describes the aging processand mechanism of sub-diffusion. This explains why sub-diffusion is notobserved for the T4 hoc⁻ phage, which is not able to adhere to mucinsand therefore never becomes ‘stuck’ during its Brownian motion.

What is the biological relevance of phage sub-diffusion in mucus?Contrary to naïve expectations that slower diffusion results inlethargic reactions, sub-diffusion can actually dramatically increasethe rate of biological encounters (Weiss & Nilsson, 2004; Höfling &Franosch, 2013). Sub-diffusive particles take longer to diffuse awayfrom their initial positions, and are more likely to retrace localregions than a freely diffusing particle (Weber et al., 2010). Thus aphage that diffuses normally could swiftly escape the mucus layer, whilethe subdiffusive phage is more likely to stay within the mucus layer fora longer period of time (Golding & Cox, 2006; Barkai et al., 2012).

Bacteriophage have Increased Adsorption with Bacterial Host in Mucus.

The search for a specific target is a ubiquitous process across biology.At the macroscopic scale we observe animals searching for food, at themicroscopic, molecules and enzymes perform site-specific searches withina cell (Sims et al., 2008; Golding & Cox, 2006). Many predators possessprior knowledge of where prey is located, and can further utilize theirsenses—sight, touch, sound—to fine-tune the forage. But a bacteriophagepossess no such knowledge or sense, and the search for a susceptiblebacterial host becomes effectively ‘blind’. Under such limitations,foraging success is largely dependent on the search strategy employed(Humphries <i>et al.</i>, 2012). It has been shown that many predators,ranging from microbes to humans, adopt a super-diffusive searchstrategy—called a Lévy flight—to optimize their success of randomsearches for food (Schuster & Levandowsky, 1996; Raichlen et al., 2014;Viswanathan et al., 1996). But when trying to apply such a searchstrategy to phage, we fall short. Phage have no locomotion, actinginstead as colloidal particles rather than as predators capable ofsuperdiffusive pursuit.

A critical and rate-limiting step in the phage life cycle is theextracellular search by a phage for a new bacterial host (Adams, 1959;Schlesinger, 1932). Phages ability to infect and reproduce within abacterial host can be entirely predicted by equations describing themovement and adsorption of inert colloidal particles under Brownianmotion (Stent, 1963; Schlesinger, 1932). These adsorption reactions areexperimentally measured by mixing a phage with its bacterial host in asuspension, and following the disappearance of free phage as theycollide with host cells, becoming irreversibly adsorbed to theirsurface, (Hyman & Abedon, 2009). These reactions are represented byadsorption constants (k), which are a function of bacterium and phagesize, rate of phage diffusion, and likelihood of attachment uponcollision, all of which are specific for a given phage-host pair (Stent,1963). The model T4 phage has a widely reported and empiricallydetermined adsorption constant of 2.4×10⁻⁹ cm³/min, which can be used totheoretically calculate phage adsorption rates at a known bacterialdensity.

We experimentally tested phage subdiffusion in mucus as an optimalsearch strategy for a bacterial host. Adsorption assays of both themucus-adherent T4 wt phage and non-adherent T4 hoc⁻ phage were performedunder low bacterial densities in both 1% mucin (wt/vol) and controlsolutions. Briefly, E. coli (1×10⁷ mL⁻¹) was mixed with a control (0%)or 1% mucin solution, followed by the addition of either T4 wt or hoc⁻phage (2×10⁵ mL⁻¹). Samples were then collected from these solutionsevery two minutes, followed by the addition of chloroform, effectivelydestroying all bacterial cells in the sample and stopping any furtherphage-host adsorptions. Adsorption assays were designed, based on the T4phage k, so that ˜22% of phages particles were theoretically adsorbed toa bacterial host over a 10-minute period. The mucus-adherent T4 wt phageadsorption to a bacterial host was significantly higher in 1% mucinsolution than in the control (FIG. 4A, Example 2, or FIG. 26A; n=6,t=−3.89, **P=0.0037). Comparatively, the adsorption of the non-adherentT4 hoc⁻ phage to a bacterial host was not significantly differentbetween the 1% mucin solution and the control (FIG. 4B, Example 2, orFIG. 26B; n=6, t=−1.33, P=0.22). Adsorption assays were repeated underhigher bacterial densities (7×10⁷ mL⁻¹), so that ˜80% of phage particleswere adsorbed to a bacterial host over a 10-minute period.Interestingly, under higher bacterial densities the increased adsorptionof T4 wt phage in 1% mucin solution was not apparent. It is known thatas the density of phage and bacterial particles increases, so does theirrandom chance of encounter (Stent, 1963). Thus we conclude that athigher phage and bacterial densities the benefit provided by asubdiffusive search strategy is masked by a high chance of randomencounters. But at sufficiently low bacterial densities, and thereforelow random chance of encounter with a bacterial host, subdiffusion inmucus provides an optimal search strategy for a bacteriophage. Thus byinteracting with mucosal surfaces, phage can mimic complex searchstrategies, becoming effectual bacterial predators.

A Conceptual Model of Phage Adherence to Mucus.

We have proposed a bacteriophage adherence to mucus (BAM) model, wherebyphage adhered to mucus, and demonstrated that this association reducedbacterial colonization and pathology (Barr, Auro, et al., 2013). Here wedesigned a life-like in vitro mucosal surface, with continual fluid-flowdynamics and mucus secretion, to test a phage therapy application of theBAM model. Only mucus-adherent phage were capable of protecting thissurface from a bacterial infection, with the non-adherent phageperforming no better than if no phage were added at all. Intriguingly,when we investigated the differences in phage detachment rates from themucus layers, we saw little difference between our two phage types,leading us to hypothesize that these adherence mechanism do notinfluence phage accumulation or residence times in a life-like mucosallayers. Instead, it is likely that the continual secretion and sloughingof the mucus layer acts to regulate phage retention times, irrespectiveof BAM mechanisms. Such dynamics were not easily addressed in ourprevious study.

At the microscopic level, phage adherence to mucus is measured by adecrease or slowing in diffusion (Barr, Auro, et al., 2013). Upondetailed investigation, phage diffusion in mucus was revealed to beanomalous over short time frames, indicative of a continuous time randomwalk (CTRW) model of subdiffusion. The CTRW model suggests that phagediffuse normally in mucus until they encounter a mucin glycoprotein,which they remain stuck to for a random period of time (Barkai et al.,2012; Ernst et al., 2014). Thus those phages stuck within the mucuslayer would not move far away from their initial positions (Metzler etal., 2009). This is an important realization. At low densities phagemake poor predators, and this slow de-correlation of phage away frommucus layers acts to maintain locally higher phage densities aroundproductive sites of infection (FIG. 5A of Example 2; or FIG. 27A). Thisaffect is further amplified by the realization that mucus-adherent phageexhibit Brownian motion at both lower and higher mucin concentrations,driving phage toward an optimal mucosal locale that likely parallelstheir bacterial host niche. Yet this explanation alone is insufficient.The mucus layer is a dynamic, ever-outwardly expanding gradient ofmucins, and phages aggregating within this optimal range will quicklyfind themselves sloughed away.

In order to visualize and understand phage subdiffusion, we must becomethe phage—now lost within a convoluted mucus network, trying to find anevasive bacterium of our own to infect (FIG. 5B of Example 2; or FIG.27B). Afloat within this environment we notice our prey, a distantbacterium barging through the mucus network, quickly squirming away.Eventually we drift into a mucin glycoprotein—a boundlessly long fiber,forming a three-dimensional spider web, covered by thousands ofhair-like glycan chains. Using the hundreds of immunoglobulin-likedomains covering our capsid, we weakly grab hold of, and then release,one of these mucin glycans. At times we hold onto many glycans, graspinghold of the mucin fiber for ever-longer periods of time, slowly driftingalong a mucin cable. This dance continues, until eventually an obliviousbacterium, stopping briefly to absorb a swath of glycans, crashes intothe mucin we are a grasp. Seizing this chance encounter, we attack thebacterium, injecting our genetic material and quickly synthesizing phageproteins and nucleic acids. Eventually, we lyse the temporary bacterialhost, releasing phage progeny back into the mucus network at higherconcentrations. Many more phage now dance along the mucin fibers,encountering significantly more bacterial hosts than those competingphage that simply drift. Very quickly our mucus-adherent phage havebecome a swarm, completely coating the mucin fibers within the immediatevicinity. Like ticks in a grass field, our phage swarm catchesprogressively more bacteria as they brush past the mucin branchesunaware.

The degree of subdiffusion can dramatically influence the time-course atwhich biological encounters take place. This mechanism has important andpreviously unrecognized implications for phage therapy. We demonstratethat only mucus-adherent phage were capable of reducing bacterialcolonization of our in vitro mucosal surface, and correlate this withincreased host adsorption as a result of subdiffusive motion. Althoughphage adherence to mucus only results in a small increased chance of anyone phage finding a bacterial host, at large abundances this biologicaleffect is dramatic. Here we apply methodologies to corroborate theconnection between subdiffusion and the biophysical world, providing amuch wanted subdiffusion experimental control (Höfling & Franosch, 2013;Saxton, 2012).

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A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of embodiments asprovided herein. Accordingly, other embodiments are within the scope ofthe following claims.

What is claimed is:
 1. A composition comprising: a plurality ofisolated, or substantially purified intact Caudovirales bacteriophagesadherent or bound to mucins or negatively charged glycans on a surfaceof: (i) an isolated or substantially purified: milk lipid, milk fatglobule (MFG) macromolecule, or a mixture thereof; (ii) an isolatedplasma membrane, or component of a plasma membrane, (iii) a hydrogel,(iv) a biocompatible crosslinked degradable thiol-ene polymer, or (v)any combination thereof, by binding or adhering to the mucins ornegatively charged glycans, wherein all of (i) to (iv) comprise andexpress on their surfaces the mucins or negatively charged glycans. 2.The composition of claim 1, wherein the Caudovirales bacteriophage areformulated per dose, or per serving, or per unit dosage at, or at atotal daily dose of: between about 10¹ and 10²⁰ plaque-forming units(PFUs), or between about 10³ and 10¹⁷ PFUs, or between about 10⁵ and10¹² PFUs, or between about 10⁷ and 10⁹ PFUs.
 3. The composition ofclaim 1, further comprising a pharmaceutically acceptable excipient. 4.The composition of claim 1, formulated for administration in vivo; orfor enteral or parenteral administration, or for ophthalmic, topical,oral, intranasal, intrarectal, intravaginal, intravenous,intra-peritoneal, intraocular, intra-placental, intra-bladder,cutaneous, intravenous (IV), intramuscular (IM), intrathecal,subcutaneous (SC), intracerebral, epidural, intracranial or rectaladministration, or by inhalation.
 5. The composition of claim 1,formulated or manufactured as or placed or formulated into: (a) atablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, asuspension, a syrup, an emulsion, a lotion, an ointment, an aerosol, aspray, a lozenge, an ophthalmic preparation, an aqueous or a sterile oran injectable solution, or a patch, wherein optionally the patch is atransdermal patch or a medicated adhesive patch, (b) an implant, adietary supplement, an ice cream, an ice, a yogurt, a cheese, an infantformula or infant dietary supplement, a pasteurized milk or milk productor milk-comprising product; or (c) a veterinary formulation or feed. 6.The composition of claim 1, further comprising, or having added to: aflavoring or a sweetening agent, an aspartamine, a stevia, monk fruit, asucralose, a saccharin, a cyclamate, a xylitol, a vanilla, an artificialvanilla or chocolate or strawberry flavor, an artificial chocolateessence, or a mixture or combination thereof; a preservative, a benzoicacid, a potassium sorbate; at least one probiotic or prebiotic, whereinoptionally the prebiotic comprises an inulin, lactulose, extracts ofartichoke, chicory root, oats, barley, various legumes, garlic, kale,beans or flacks or an herb, at least one congealing agent, whereinoptionally the congealing agent comprises an arrowroot or a plantstarch, a powdered flour, a powdered potato or potato starch, anabsorbant polymer, an ABSORBABLE MODIFIED POLYMER® (AMP®), a corn flouror a corn starch; at least one anti-inflammatory agent, whereinoptionally the at least one inflammatory agent comprises or is an NSAID,a 4 or a 5-amino-salicylate, an olsalazine, a mesalazine, asulfasalazine, a balsalazide, or an equivalent thereof or a combinationthereof; or an additive selected from one or more of a saline, a media,a defoaming agent, a surfactant agent, a lubricant, an acid neutralizer,a marker, a cell marker, a drug, an antibiotic, a contrast agent, adispersal agent, a buffer or a buffering agent or a pharmaceuticallyacceptable excipient, a sweetening agent, a debittering agent, aflavoring agent, a pH stabilizer, an acidifying agent, a preservative, adesweetening agent and/or coloring agent, vitamin, mineral and/ordietary supplement, an antacid or a prebiotic nutrient, and optionallythe buffer or a buffering agent or the pharmaceutically acceptableexcipient comprises an inorganic salt, a citric acid, a sodium chloride,a potassium chloride, a sodium sulfate, a potassium nitrate, a sodiumphosphate monobasic, a sodium phosphate dibasic or combinations thereof,and optionally the antacid comprises a calcium carbonate, a magnesiumhydroxide, a magnesium oxide, a magnesium carbonate, an aluminumhydroxide, a sodium bicarbonate or a dihydroxyaluminum sodium carbonate;or any combination thereof.
 7. The composition of claim 1, wherein thecomposition is isolated, treated, formulated or prepared as a delayed orgradual enteric release composition.
 8. The composition of claim 1,wherein the isolated, or substantially purified milk fat globule (MFG)macromolecule has a particle size between about: 1 nm to 100 nm, 100 nmto 1000 nm, 1 um to 100 um, or 100 um to 1000 um.
 9. The composition ofclaim 1, wherein the isolated, or substantially purified milk fatglobule (MFG) macromolecule comprises between about 0.01% to 1%, orbetween about 1% to 20% of the composition.
 10. A delivery vehicle, aproduct of manufacture, a container, a syringe, device or an implant,comprising: the composition of claim
 1. 11. A liquid, a suspension, agel, a geltab, a semisolid, a tablet, a sachet, a lozenge or a capsule,a freeze-dried composition, an infant formula, or an enteral orparenteral formulation, comprising: the composition of claim
 1. 12. Thecomposition of claim 1, wherein the isolated, or substantially purifiedmilk lipid or milk fat globule (MFG) macromolecule is derived from amammalian milk.
 13. The composition of claim 1, wherein the plasmamembrane, or component of a plasma membrane: (a) is reconstituted as aglobular particle of a specific size range, and optionally the globularparticle has a size of between about: 1 nm to 100 nm, 100 nm to 1000 nm,1 um to 100 um, or 100 um to 1000 um; or (b) is a bacterial plasmamembrane, or component of a bacterial plasma membrane.
 14. Thecomposition of claim 3, wherein the composition comprises: anantibiotic, a microorganism, and optionally the microorganism is abacteria or a yeast, and optionally the bacteria is a probioticbacteria, or a probiotic, a prebiotic, a postbiotic, or a synbiotic,wherein optionally the prebiotic comprises a nondigestibleoligosaccharide, and optionally the nondigestible oligosaccharidecomprise an inulin, a fructan, a xylose, a maltose, a mannose, or afructooligosaccharide, and optionally the probiotic bacteria comprise: aBacillus or a Lactobacillus; or, a Bacillus coagulans, a Lactobacillusacidophilus, a Lactobacillus plantarum or a Lactobacillus rhamnosus, andoptionally the yeast comprises a Saccharomyces; or a Saccharomycesboulardii.
 15. The composition of claim 1, wherein the plurality ofisolated, or substantially purified intact Caudovirales bacteriophagesare formulated or comprise a payload to treat, inhibit an infection, andoptionally the infection is a microbial or a bacterial infection, andoptionally the intact bacteriophages are bacteriocidal or bacteriostaticto gram negative bacteria or gram positive bacteria, and optionally thebacteria or infection is or is caused by an MSRA infection, aStaphylococcus, a Staphylococcus aureus, a Clostridium, or a Clostridiumdifficile, Escherichia coli, a Shigella, a Salmonella, a Campylobacter,a Cholerae Chloerae, a Bacillus, or a Yersinia.
 16. The composition ofclaim 1, wherein the plurality of isolated, or substantially purifiedCaudovirales bacteriophages are adherent to the surface of an isolatedor substantially purified: milk lipid, milk fat globule (MFG)macromolecule, or a mixture thereof.
 17. The composition of claim 1,wherein the plurality of isolated, or substantially purifiedbacteriophages are adherent to the surface of a plasma membrane.
 18. Thecomposition of claim 1, wherein the plurality of isolated, orsubstantially purified bacteriophages are adherent to the surface of ahydrogel.
 19. The composition of claim 1, wherein the plurality ofisolated, or substantially purified bacteriophages are adherent to thesurface of a biocompatible crosslinked degradable thiol-ene polymer. 20.The composition of claim 12, wherein the mammalian milk is a human milk.21. The composition of claim 7, wherein the formulation comprises agastro-resistant coating designed to dissolve at a pH of 7 in theterminal ileum.
 22. The composition of claim 7, wherein the compositionis coated with an acrylic based resin or equivalent, and optionally theacrylic based resin or equivalent comprises a poly(meth)acrylate, amethacrylic acid copolymer B, or NF, and optionally the acrylic basedresin or equivalent comprises EUDRAGIT S™, which dissolves at pH 7 orgreater, and optionally the acrylic based resin or equivalent comprisesa multimatrix (MMX) formulation.
 23. The composition of claim 1, whereinthe composition is manufactured or formulated as a food or a feed, adrink, a nutraceutical, a pharmaceutical formulation, a pharmaceuticalor a pharmaceutical preparation.